Complete genome sequence of the methanogen methanobrevibacter ruminantium

ABSTRACT

The present invention includes the complete genome sequence for the methanogen,  Methanobrevibacter ruminan - tium , including polynucleotides which encode  M. ruminantium  polypeptides or peptides, as well as polynucleotides from non-coding regions. Also included are the encoded  M. ruminantium  polypeptides and peptides, and antibodies directed to these peptides or polypeptides, in addition to expression vectors and host cells for producing these peptides, polypeptides, polynucleotides, and antibodies. The invention further includes methods and compositions for detecting, targeting, and inhibiting microbial cells, especially methanogen cells such as  M. ruminantium  cells, using one or more of the disclosed peptides, polypeptides, polynucleotides, antibodies, expression vectors, and host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/285,008, filed on Feb. 25, 2019, which is a divisional of U.S. patentapplication Ser. No. 15/213,596, filed on Jul. 19, 2016, now U.S. Pat.No. 10,314,895, which is a Continuation of U.S. patent application Ser.No. 13/392,075, filed May 7, 2012, now U.S. Pat. No. 9,441,016, which isa 35 U.S.C. 371 National Phase of PCT Application No. PCT/NZ2010/000169,filed Aug. 27, 2010, which claims priority to Provisional PatentApplication No. 61/237,296, filed Aug. 27, 2009, and New ZealandProvisional Patent Application No. 579292, filed Aug. 27, 2009, thedisclosures of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention encompasses the complete genome sequence for themethanogen, Methanobrevibacter ruminantium. The invention encompassespolynucleotides which encode M. ruminantium polypeptides or peptides, aswell as polynucleotides from non-coding and intergenic regions. Alsoencompassed are the encoded M. ruminantium polypeptides and peptides,and antibodies directed to these peptides or polypeptides. The inventionalso encompasses expression vectors and host cells for producing thesepeptides, polypeptides, polynucleotides, and antibodies. The inventionfurther encompasses methods and compositions for detecting, targeting,and inhibiting microbial cells, especially methanogen cells such as M.ruminantium cells, using one or more of the disclosed peptides,polypeptides, polynucleotides, antibodies, expression vectors, and hostcells.

BACKGROUND OF THE INVENTION

Global surface temperatures are predicted to increase between 1.1° C. to6.4° C. during the twenty-first century primarily due to increasedlevels of greenhouse gases (GHGs) in the atmosphere (Solomon et al.,2007). Methane (CH₄) is a particularly potent GHG, having a globalwarming potential 21 times that of carbon dioxide (CO₂) (IPCC, 2007) andaccounts for 16% of total global GHG emissions (Scheehle & Kruger,2006). Methane from agriculture represents around 40% of the emissionsproduced by human-related activities, the single largest source of whichis from enteric fermentation in livestock, mainly from ruminant animals(Steinfeld et al., 2006). The worldwide demand for meat and milk ispredicted to double by 2050 (Food and Agriculture Organization of theUnited Nations (FAO), 2008) and ruminant-based agricultural activitiesare expected to continue to be an important contributor to global CH₄emissions. Therefore, reducing CH₄ emissions from ruminants will beimportant in meeting international commitments under the Kyoto Protocoland also in ensuring the long-term sustainability of ruminant-basedagriculture. Moreover, as CH₄ production in the rumen accounts for 2-12%of the ingested energy (Johnson & Johnson, 1995), it is predicted thatreducing CH₄ emissions from ruminants will also make more energyavailable to the animal and therefore enhance their productivity.Ruminant animals are particularly important to agriculture in NewZealand (NZ), producing a third of NZ's commodity exports (StatisticsNew Zealand, 2008) and making up a large proportion of theinternationally traded lamb and milk products (Leslie et al., 2008).Consequently, NZ has an unusual GHG emission profile, with ruminant CH₄emissions accounting for 31% of NZ's total GHG emissions (Ministry forthe Environment, 2007).

Methane is formed in the fore-stomach (reticulorumen, or more commonlyknown as the rumen) by methanogens, a subgroup of the Archaea. Duringnormal rumen function, plant material is broken down by fibre-degradingmicroorganisms and fermented mainly to volatile fatty acids (VFAs),ammonia, hydrogen (H₂) and CO₂. Rumen methanogens principally use H₂ toreduce CO₂ to CH₄ in a series of reactions that are coupled to ATPsynthesis. The rumen harbours a variety of different methanogen species,but analyses of archaeal small subunit ribosomal RNA genes from rumensamples of ruminants on differing diets around the world suggest themajority fall into three main groups: Methanobrevibacter,Methanomicrobium and a large, as yet uncultured, group of rumen archaeareferred to as rumen cluster C (Janssen & Kirs, 2008). Sequencesaffiliated with Methanobrevibacter dominate, on average accounting for61.6% of rumen archaea, with sequences associated with M. gottschalkii(33.6%) and M. ruminantium (27.3%) being most prominent.

Attempts have been made to inhibit the action of methanogens in therumen using a variety of interventions but most have failed, or met withonly limited success, due to low efficacy, poor selectivity, toxicity ofcompounds against the host, or build up of resistance to anti-methanogencompounds (McAllister & Newbold, 2008). Currently there are fewpractical methane reduction technologies available for housed ruminantanimals, and no effective technologies for grazing animals. Methaneinterventions should ideally target features that are conserved acrossall rumen methanogens, so that no unaffected methanogens can fill thevacated niche. Interventions should also be specific for methanogensonly, such that other rumen microbes continue their normal digestivefunctions. Whole genome sequencing allows the definition of gene targetsthat are both conserved and specific to rumen methanogens. It is not yetpossible to obtain genome sequences of all methanogen groups present inthe rumen as some are yet to be cultivated, and a rumen methanogen“metagenome” is prevented by the inability of current sequencingtechnologies to reassemble complete genomes from complex microbialecosystems. Therefore, sequencing the genomes of individual rumenmethanogens currently in culture is a critical step in developing CH₄mitigation technologies for ruminant animals.

SUMMARY OF THE INVENTION

Here we report the genome sequence of M. ruminantium M1T (DSM 1093), thefirst rumen methanogen genome to be completely sequenced. We haveincluded a particular emphasis on identifying targets for entericmethane mitigation technologies focusing on vaccine development andanti-methanogen drug leads.

The invention thus features the complete genome sequence for themethanogen, Methanobrevibacter ruminantium. The invention features, inparticular, isolated peptides, polypeptides, and polynucleotides of M.ruminantium, as well as expression vectors, host cells, and antibodies,and methods of use thereof, as described in detail herein.

The invention specifically features an isolated peptide comprising, forexample, at least a fragment of one amino acid sequence selected fromthe group consisting of SEQ ID NO: 5867-7584. In a particular aspect,the peptide comprises at least a fragment of an amino acid sequence ofany one of SEQ ID NO: 5867-7584. In a further aspect, the peptidecomprises at least a fragment of an amino acid sequence of any one ofSEQ ID NO: 5867-7584. In another aspect, the peptide is a fragment, forexample, comprising at least one amino acid sequence encompassing anextracellular domain of any one of SEQ ID NO: 5867-7584.

The invention specifically features an isolated polypeptide comprising,for example, at least one amino acid sequence selected from the groupconsisting of SEQ ID NO: 5867-7584. In a particular aspect, thepolypeptide comprises the amino acid sequence of any one of SEQ ID NO:5867-7584. In a further aspect, the polypeptide comprises the amino acidsequence of any one of SEQ ID NO: 5867-7584. In another aspect, thepolypeptide is a fragment, for example, comprising at least one aminoacid sequence encompassing an extracellular domain of any one of SEQ IDNO: 5867-7584.

The invention additionally features an isolated polynucleotidecomprising a coding sequence for at least one peptide. In one aspect,the polynucleotide comprises a coding sequence for at least a fragmentof an amino acid sequence selected from the group consisting of SEQ IDNO: 5867-7584. In a particular aspect, the polynucleotide comprises acoding sequence for at least a fragment of any one of SEQ ID NO:5867-7584. In a further aspect, the polynucleotide comprises a codingsequence for at least a fragment of any one of SEQ ID NO: 5867-7584. Inanother aspect, the polynucleotide comprises a fragment of a codingsequence, for example, least one amino acid sequence encompassing anextracellular domain of any one of SEQ ID NO: 5867-7584.

The invention additionally features an isolated polynucleotidecomprising a coding sequence for at least one polypeptide. In oneaspect, the polynucleotide comprises a coding sequence for at least oneamino acid sequence selected from the group consisting of SEQ ID NO:5867-7584. In a particular aspect, the polynucleotide comprises a codingsequence for any one of SEQ ID NO: 5867-7584. In a further aspect, thepolynucleotide comprises a coding sequence for any one of SEQ ID NO:5867-7584. In another aspect, the polynucleotide comprises a fragment ofa coding sequence, for example, least one amino acid sequenceencompassing an extracellular domain of any one of SEQ ID NO: 5867-7584.

In an additional aspect, the invention features an isolatedpolynucleotide comprising, for example, a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1-1718. In a particular aspect,the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:1-1718. In another aspect, the polynucleotide is a fragment or anoligonucleotide comprising, for example, the nucleic acid sequenceencompassing an extracellular domain as encoded by any one of SEQ ID NO:1-1718. In addition, the invention encompasses an isolatedpolynucleotide, or fragment thereof, which hybridizes to any one of thenucleic acid sequences of SEQ ID NO: 1-1718. The invention furtherencompasses an isolated polynucleotide comprising the complement,reverse complement, reverse sequence, or fragments thereof, of any oneof the nucleic acid sequences.

The invention features an expression vector comprising a polynucleotideof the invention. In one aspect, the expression vector comprises acoding sequence for at least a fragment of an amino acid sequenceselected from the group consisting of SEQ ID NO: 5867-7584. In aparticular aspect, the expression vector comprises a coding sequence forat least a fragment of at least one of SEQ ID NO: 5867-7584. In afurther aspect, the expression vector comprises a coding sequence for atleast one amino acid sequence of at least one of SEQ ID NO: 5867-7584.In another aspect, the expression vector comprises a coding sequence forat least one amino acid sequence encompassing an extracellular domain ofany one of SEQ ID NO: 5867-7584.

The invention also features a host cell, for example, a microbial hostcell, comprising at least one expression vector.

The invention specifically features an antibody directed to a peptide,polypeptide, or polynucleotide as disclosed herein. In certain aspects,the antibody is directed to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 5867-7584. In alternate aspects, theantibody is directed to at least a fragment of a polypeptide sequenceselected from the group consisting of SEQ ID NO: 5867-7584. In aparticular aspect, the antibody binds to at least a fragment of thepeptide sequence of any one of SEQ ID NO: 5867-7584. In a furtheraspect, the antibody binds to at least a fragment of the polypeptidesequence of any one of SEQ ID NO: 5867-7584. In an alternate aspect, theantibody binds to at least a fragment of a peptide or polypeptideencompassing an extracellular domain of any one of SEQ ID NO: 5867-7584.In another aspect, the antibody includes one or more fusions orconjugates with at least one cell inhibitor, for example,anti-methanogenesis compounds (e.g., bromoethanesulphonic acid),antibodies and antibody fragments, lytic enzymes, peptide nucleic acids,antimicrobial peptides, and other antibiotics as described in detailherein.

The invention additionally features modified peptides or polypeptides,e.g., for at least one of SEQ ID NO: 5867-7584, including biologicallyactive alterations, fragments, variants, and derivatives, describedherein. Also featured are polynucleotides encoding these modifiedpeptides or polypeptides, as well as alterations, fragments, variants,and derivatives of the disclosed polynucleotides; antibodies raisedusing these modified peptides, polypeptides, or polynucleotides;expression vectors comprising these polynucleotides; and host cellscomprising these vectors. Further featured are modified antibodies,including biologically active alterations, fragments, variants, andderivatives, described herein. In specific aspects, the compositions andmethods of the invention employ these modified peptides, polypeptides,polynucleotides, antibodies, or corresponding expression vectors or hostcells.

The invention features a composition comprising an isolated peptide orpolypeptide, e.g., at least one of SEQ ID NO: 5867-7584. Also featuredis a composition comprising an isolated polynucleotide, e.g., at leastone of SEQ ID NO: 1-1718. The invention additionally features acomposition comprising an antibody, e.g., directed to a peptide,polypeptide, or polynucleotide sequence disclosed herein. Furtherfeatured is a composition that includes an expression vector, or hostcell comprising an expression vector, in accordance with the invention.The composition can include any one of the biologically activealterations, fragments, variants, and derivatives described herein. Thecompositions can include at least one cell inhibitor (e.g., as a fusionor conjugate), and can be formulated, for example, as pharmaceuticalcompositions, in particular, vaccine compositions.

The invention also features a composition of the invention as part of akit for targeting and/or inhibiting microbial cells, especiallymethanogen cells, in accordance with the disclosed methods. The kitscomprise: a) at least one composition as set out herein; and b)optionally, instructions for use, for example, in targeting cells orinhibiting cell growth or replication for methanogens or other microbes.

The invention also features a method for producing a peptide orpolypeptide, e.g., at least a fragment of any one of SEQ ID NO:5867-7584, the method comprising: a) culturing an expression vector orhost cell comprising an expression vector, which comprises at least partof a coding sequence for at least one peptide or polypeptide underconditions suitable for the expression of the peptide or polypeptide;and b) recovering the peptide or polypeptide from the culture. Inparticular aspects, the peptide or polypeptide comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NO:5867-7584, or modified sequences thereof.

The invention also features a method for producing an antibody, e.g.,directed to at least a fragment of any one of SEQ ID NO: 5867-7584, themethod comprising: a) culturing an expression vector or host cellcomprising an expression vector, which comprises at least part of acoding sequence for at least one antibody or antibody fragment underconditions suitable for the expression of the antibody or antibodyfragment; and b) recovering the amino acid sequence from the culture. Inparticular aspects, the antibody or antibody fragment is directed to atleast one amino acid sequence selected from the group consisting of SEQID NO: 5867-7584, or modified sequences thereof. In an alternate aspect,the antibody is produced by administration to a host animal, asdescribed in detail herein.

The invention additionally features a method for producing an antibody,e.g., directed to at least a fragment of any one of SEQ ID NO:5867-7584, which comprises a fusion or conjugate with at least one cellinhibitor. Such method comprises: a) culturing an expression vector orhost cell comprising an expression vector, which comprises a codingsequence for at least one antibody or antibody fragment under conditionssuitable for the expression of the antibody or antibody fragment; b)forming a fusion or conjugate to the antibody or antibody fragment(e.g., by expression of the fused sequence or chemical conjugation tothe cell inhibitor); and c) recovering the fusion or conjugate.

In particular aspects, the antibody is directed to at least a fragmentof any one of SEQ ID NO: 5867-7584, or modified sequences thereof. Infurther aspects, the inhibitor is selected from anti-methanogenesiscompounds (e.g., bromoethanesulphonic acid), antibodies and antibodyfragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides,and other antibiotics as described in detail herein. In an alternateaspect, the antibody is produced by administration to a host animal andthen conjugated, as described in detail herein.

In addition, the invention features a method of inhibiting (e.g.,inhibiting growth or replication) of a microbial cell, in particular, amethanogen cell, comprising: contacting the cell with antibody orantibody fragment, e.g., directed to at least a fragment of any one ofSEQ ID NO: 5867-7584, or an antibody fusion or conjugate, or anymodified antibody. As another method, the cell is inhibited byadministration of a vaccine composition as described in detail herein.

The invention further features a method of inhibiting (e.g., inhibitinggrowth or replication) of a microbial cell, in particular, a methanogencell, comprising: a) optionally, producing or isolating at least oneantibody as disclosed herein; and b) contacting the cell with theantibody. In a particular aspect, the antibody is directed to at least afragment of any one of SEQ ID NO: 5867-7584, or a modified sequencethereof. In certain aspects, the antibody further comprises at least onecell inhibitor, attached, for example, as a fusion or conjugate. Inother aspects, the antibody is administered to a subject as acomposition, e.g., a vaccine composition.

Additionally, the invention features a method of inhibiting (e.g.,inhibiting growth or replication) of a microbial cell, in particular, amethanogen cell, comprising: a) optionally, producing or isolating atleast one peptide or polypeptide as disclosed herein; and b)administering the peptide or polypeptide to a subject to induce animmune response thereto. In a particular aspect, the peptide orpolypeptide comprises at least one amino acid sequence selected from thegroup consisting of SEQ ID NO: 5867-7584, or a modified sequencethereof. In other aspects, the peptide or polypeptide is administered toa subject as a composition, e.g., a vaccine composition.

The invention furthermore features a method of detecting and/ormeasuring the levels of a polypeptide, in particular, a cell surfacepolypeptide, or corresponding peptides or polynucleotides,comprising: 1) contacting a sample from a subject with an antibodydirected to the polypeptide (e.g., at least a fragment of any one of SEQID NO: 5867-7584, or a modified sequence thereof), or a correspondingpeptide or polynucleotide (e.g., at least a fragment of one of SEQ IDNO: 1-1718, or a modified sequence thereof); and 2) determining thepresence or levels of the antibody complex formed with the correspondingpolypeptide, peptide, or polynucleotide in the sample. Such methods canalso be used for detecting and/or measuring the levels of a microbialcell, in particular, a methanogen cell.

The invention also features a method of detecting and/or measuring thelevels of a polynucleotide, in particular, a polynucleotide encoding acell surface component, comprising: 1) contacting a sample from asubject with a complementary polynucleotide (e.g., a sequencecomplementary to at least a fragment of any one of SEQ ID NO: 1-1718, ora modified sequence thereof); and 2) determining the presence or levelsof the hybridization complex formed with the polynucleotide in thesample. Such methods can also be used for detecting and/or measuring thelevels of a microbial cell, in particular, a methanogen cell.

In particular aspects, the methods of the invention utilize in vivo orin vitro expression components. In other aspects, the methods employpeptides, polypeptides, polynucleotides, or antibodies produced byrecombinant, synthetic, or semi-synthetic means, or by endogenous means.

Other aspects and embodiments of the invention are described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is described with reference to specific embodimentsthereof and with reference to the figures.

FIG. 1: Chemogenomic and vaccine gene targets within M1. The number ofgenes identified by each analysis is shown in the Venn diagram and aselection of the gene targets are summarized in the tables grouped byfunctional category (a) Chemogenomic gene targets were defined byidentification of genes that occurred across three separate analyses;the Functional Genome Distribution (FGD), Differential BLAST analysis(DBA) and metabolic profiling. (b) Vaccine target genes were defined asdescribed and discussed below. TMH, transmembrane helices, SP, signalpeptide.

FIG. 2: GC analysis. Base-pair scale (outer circle), G+C content (middlecircle) and GC skew (inner circle, (G−C/G+C), darker shade indicatesvalues >1, lighter shade <1. Genomes of the Methanobacteriales orderdisplay a DNA skew similar to bacterial chromosomes. In M. ruminantiumM1 the origin of replication (oriC) was identified as being immediatelyupstream of the Cdc6-1 gene (mru0001) based on GC skew analysis andhomology to the origin of replication experimentally verified for M.thermoautotrophicus (Reymond et al., 2004). As with otherMethanobacteriales genomes, M. ruminantium M1 contains a second Cdc6homolog (mru0423). It also contains a truncated third Cdc6 homologwithin the prophage sequence.

FIG. 3: PROmer alignments (Delcher et al., 2003) of M. ruminantium M1against complete Methanobacteriales genomes. Whenever the two sequencesagree, a shaded line or dot is plotted. The forward matches aredisplayed in the lighter shade, while the reverse matches are plotted inthe darker shade. Where the two sequences were perfectly identical, asingle lighter line would extend from the bottom left to the top right.An X-shape pattern is visible is all three synteny plots indicatingmoderately diverged Methanobacteriales genomes. It has been proposedthat the X-pattern is generated by symmetric chromosomal inversionsaround the origin of replication (Emanuelsson, 2007). Units displayed inbase-pairs.

FIG. 4-1-4-4: Consensus sequence of forty-four C-terminal regions (200amino acids) from adhesin-like proteins of M. ruminantium M1 (A).LogoBar display of this consensus. In both figures region of homology toBig_1 domain (PfamO2369) is highlighted in grey (B).

FIG. 5: Proposed biosynthetic pathway for pseudomurein in M1 (Kandlerand König H, 1998; König et al., 1994). The disaccharide backbone of M.ruminantium M1 pseudomurein consists of N-acetylgalactosamine (GalNAc)and N-acetyltalosaminuronic acid (TalNAc) in a β(1-3) linkage. TalNachas not been detected as a monomer and it is believed to be formedduring the synthesis of the disaccharide probably by epimerization andoxidation of UDP-GalNAc (Step 1). Synthesis of the pentapeptide involvedin crosslinking is believed to start with UDP-glutamic acid followed bystepwise addition of L-amino acids (Step 2). The amino acids found inthe pentapeptide are usually alanine, lysine (Lys) and glutamic acid(Glu), but M. ruminantium M1 is reported to contain threonine (Thr)instead of alanine (Kandler and König, 1978). The UDP activatedpentapetide is linked to the disaccharide to give a UDP-disaccharidepentapeptide (Step 3) which is subsequently translocated to the membranevia covalent bond formation with a membrane embedded undecaprenylphosphate (Step 4). Following their intracellular biosynthesis thepseudomurein repeating units must be exported and assembled. Homologs ofthe E. coli peptidoglycan lipid II flippase (MurJ) have been reportedfor pseudomurein producing methanogens (Ruiz, 2008; Step 5), but thereare no genes similar to the penicillin binding proteins that carry outthe transglycosylation (Step 6) and transpeptidation reactions inbacterial peptidoglycan assembly. Peptide crosslinking of pseudomureinrequires removal of a terminal residue of one peptide and linkage from aglutamic acid to the lysine of an adjacent peptide (Step 7), and isprobably carried out by transglutaminases. None of the enzymes involvedin pseudomurein biosynthesis have been characterized, but analysis ofthe genome sequence has suggested candidates to carry out several of thesteps. Several of these have homologs only in those methanogens withpseudomurein-containing cell walls. Two other transmembrane proteins ofunknown function (mru1585 and mru1635) are also only found inpseudomurein-containing species.

FIG. 6: (a) PFGE of genomic DNA from M1. Lane 1, A ladder (New EnglandBiolabs); Lane 2, ApaI/BssHII double digest; Lane 3, ApaI digest; Lane4, Mlul digest; Lane 5, Sizes of MluI fragments. The bands in the Aladder are multiples of 48.5 kb. (b) In silico restriction map of the M1chromosome showing the position and fragment size of the Mlul digest.

FIG. 7: Gene organisation of three clusters proposed to be involved insecondary metabolite metabolism in M1. Cluster1. Mru0068 is predicted toencode two non-ribosomal peptide synthetase (NRPS) modules, eachcontaining condensation, adenylation and thiolation domains. Thepresence of a condensation domain in the first module is oftenassociated with NRPSs that make N-acylated peptides (Steller et al.,1999). The second module is followed by a terminal thioester reductasedomain which is thought to release the peptide from the final thiolationdomain. Mru0068 is surrounded by genes that encode two serinephosphatases (mru0066, mru0071), an anti-sigma factor antagonist(mru0067) and a MatE efflux family protein (mru0069) which are likely tobe involved in environment sensing, regulating NRPS expression andexport of the NRP, respectively. Cluster2. The second NRPS gene(mru0351) contains 4 modules and a C-terminal thioester reductasedomain. Immediately downstream of mru0351 is another MatE efflux familyprotein (mru0352), presumably involved in the efflux of the NRP.Cluster3. A small cluster of genes elsewhere in the genome(mru0513-0516) appears to encode NRPS-associated functions. The clusterincludes a 4′-phosphopantetheinyl transferase (mru0514) which primesNRPSs by adding a phosphopantetheinyl group to a conserved serine withinthe thiolation domain, an acyltransferase (mru0512) possibly involved inNRP acylation, a serine phosphatase (mru0515), an anti-sigma factorantagonist (mru0513), and an anti-sigma regulatory factorserine/threonine protein kinase (mru0516) that may function in sensingthe environment and NRPS regulation. Although the products of each NRPSare unknown, an analysis of adenylation domain amino acid sequences byNRPSpredictor (Rausch et al., 2005) predicts 10 residues which areimportant for substrate binding and catalysis.

FIG. 8: Effect of the lytic enzyme PeiR on M1 growth in vitro. (A)Addition of PeiR to growing cultures at 73 h resulted in a dramatic dropin culture density, indicative of cell lysis. At a low concentration ofPeiR (final concentration of 2.5 mg per litre), the cultures were ableto recover, indicated by the increase in culture density after 100 h,and (B) by production of methane at levels similar to that of culturesreceiving no PeiR. Addition of higher concentrations of PeiR (7.5 and22.5 mg per litre) resulted in a lasting effect, with (A) no subsequentrecovery of growth and (B) a reduced methane yield. Chloroform, a knownpotent inhibitor of methanogens, resulted in a similarly reduced methaneyield (B), but the decrease in culture density was less (A), as expectedsince it halts metabolism rather than lysing cells. PeiR was added to 10ml cultures in 0.1 ml of buffer. The buffer alone had no effect. Thesymbols (A) and solid bars (B) are means of 3 replicates, and the thinvertical bars represent one standard error on either side of the mean.

FIG. 9: Observation of interspecies interactions between M. ruminantiumM1 and B. proteoclasticus B316^(T). Graph displays growth rate of M1 inco-culture with B316. Microscopic images taken at 2, 8 and 12 h postinoculation of B316 (lighter, rod-shaped organism) into BY+(+0.2% xylan)media containing a mid-exponential M1 culture (darker, short ovoidrod-shaped organism). Growth as determined by Thoma slide enumeration,is shown along with sampling time.

FIG. 10: Genome atlas of M. ruminantium M1. The circle was created usingGenewiz (Jensen et al., 1999) and in house developed software. Theright-hand legend describes the single circles in thetop-down-outermost-innermost direction. Outermost 1^(st) ring:Differential Blast Analysis between the non-redundant (nr) database(Ring 3) and a custom methanogen database (Ring 2). Regions inmedium-dark shading indicate protein sequences highly conserved betweenM. ruminantium and at methanogens but not found in the nr database.Regions in darker shading indicate protein sequences conserved betweenM. ruminantium and the nr database but not present in other methanogensgenomes. 2^(nd) ring: gapped BlastP results using a custom methanogendatabase consisting of publicly accessible genome project sequences(Table 10), 3^(rd) ring: gapped BlastP results using the non-redundantdatabase minus published methanogen genome project sequences. In bothrings, regions in medium-dark shading represent unique proteins in M.ruminantium, whereas highly conserved features are shown in darkershading. The degree of colour saturation corresponds to the level ofsimilarity. 4^(th) ring: G+C content deviation: medium shadinghighlights low-GC regions, light shading high-GC islands. Annotationrings 5 and 6 indicate absolute position of functional features asindicated. 7^(th) ring: ORF orientation. ORFs in sense orientation(ORF+) are shown in dark shading; ORFs oriented in antisense direction(ORF−) are shown in medium-dark shading. 8^(th) ring: prediction ofmembrane bound and cell surface proteins. White: no transmembranehelices (TMH) were identified, Black: ORFs with at least one TMH,Medium-dark shading: ORFs predicted to encompass a Signal Peptidesequence and Medium shading: ORFs predicted to incorporate both TMH andSignalP domains. 9^(th) ring: COG classification. COG families wereassembled into 5 major groups: information storage and processing (lightshading); cellular processes and signalling (medium shading); metabolism(light-medium shading); poorly characterized (dark shading); and ORFswith uncharacterized COGs or no COG assignment (grey). 10^(th) ring:GC-skew. Innermost ring: genome size (Mb). Selected featuresrepresenting single ORFs are shown outside of circle 1 with barsindicating their absolute size. Origin and terminus of DNA replicationare identified in light-medium shading and dark-medium shading,respectively.

FIG. 11: Methanogenesis pathway. The predicted pathway of methaneformation in M1 based on the scheme of Thauer et al. (Thauer et al.,2008) for methanogens without cytochromes. The pathway is divided intothree partitions; capture of reductant (left side, medium shadedbackground), reduction of CO₂ (centre, lighter shaded background) andenergy conservation (right side, darker shaded background). The mainreactions are indicated by thick arrows and enzymes catalysing each stepare coloured green. Cofactor participation is indicated with thinarrows. Membrane located proteins are coloured light brown and potentialvaccine and chemogenomic targets are labelled with a circled V or Crespectively. Small upwards arrows signify up-regulated genes duringco-culture with Butyrivibrio proteoclasticus. Full gene names andcorresponding locus tag numbers can be found in Table 9, below. H₄MPT;tetrahydromethanopterin; MF, methanofuran; F₄₂₀, co-factor F₄₂₀oxidised; F₄₂₀H₂, co-factor F₄₂₀ reduced; Fed_(ox))?, unknown oxidisedferredoxin; Fed(red)?, unknown reduced ferredoxin; HSCoM, reducedcoenzyme M; HSCoB, reduced coenzyme B, CoBS-SCoM, coenzyme B-coenzyme Mheterodisulphide; NADP+, nicotinamide adenosine dinucleotide phosphatenon-reduced; NADPH, nicotinamide adenosine dinucleotide phosphatereduced.

FIG. 12: Cell envelope topography of M1. Ultrastructural studies of M1(Zeikus & Bowen, 1975; Miller, 2001) show that the cell wall is composedof three layers and is comparable to the organization seen in Grampositive bacteria (Graham & Beveridge, 1994). The three layers can bedescribed as: (1) a thin electron-dense inner layer composed ofcompacted newly synthesised pseudomurein, (2) a thickerless-electron-dense middle layer which is also composed of pseudomurein,and (3) a rough irregular outer layer that is distal to the pseudomureinlayers and assumed to be composed of cell wall glycopolymers, wallassociated proteins and possibly other components. Representativeadhesin-like proteins with different cell-anchoring domains are shown.The number of these proteins predicted in the M1 genome is shown inbrackets. OT, oligosaccharyl transferase; Sec, Sec protein secretionpathway; PMBR, pseudomurein binding repeat (PF09373); M1-C, M1adhesin-like protein conserved C-terminal domain.

FIG. 13: Functional Genome Distribution (FGD) of 26 methanogen genomes.Publicly available complete genomes were downloaded in GenBank formatwere possible. Publicly available draft phase genomes were downloaded inFASTA format, concatenated using a universal spacer-stop-spacer sequence(TTAGTTAGTTAG; SEQ ID NO: 7585) and automatically annotated usingGAMOLA. Predicted ORFeomes of all genomes were subjected to an FGDanalysis and the resulting distance matrix was imported into MEGA4(Samuel et al., 2007). The functional distribution was visualised usingthe UPGMA method (Boekhorst et al., 2005). The optimal tree with the sumof branch length=49.7 is shown. The tree is drawn to scale, with branchlengths in the same units as those of the functional distances used toinfer the distribution tree. Accession numbers for individual genomescan be found in Table 10, below.

FIG. 14: Stimulation of growth of M1 by alcohols. The inclusion of (A)20 mM methanol or (B) 5 or 10 mM ethanol when M1 was grown on H2resulted in an increase in culture density (measured as OD600 nm)compared to cultures grown on H2 alone. H2 was added once only, at thetime of inoculation, by gassing the cultures with H2+CO2 (4:1) to 180kPa overpressure. Higher concentrations of ethanol (20 mM) resulted insome inhibition of growth (not shown), and there was no stimulation byisopropanol (5 to 20 mM; not shown). No growth occurred when cultureswere supplemented with methanol (A), ethanol (B), or isopropanol (notshown) when no H2 was added, and no methane was formed by thosecultures. The symbols in panel are means of 4 replicates, and the thinvertical bars in panel (A) represent one standard error on either sideof the mean. Error bars are omitted from panel (B) for the sake ofclarity.

FIG. 15: Distribution of genes in the predicted ORFeomes of members ofthe Methanobacteriales classified according to functional categories inthe archaeal COG database (Makarova et al., 2007).

FIG. 16: BLAST Heat Map depicting BLAST-result distribution across theM. ruminantium M1. ORFeome. In both figures, the X-axis (horizontalaxis) shows all genera with at least 500 and 250 Blast hits throughoutthe ORFeome, respectively. Genera are phylogenetically sorted based on asemi-dynamically re-parsed phylogenetic tree obtained from the RibosomalDatabase Project II (RDP II) (hypertext transferprotocol://rdp.cme.msu.edu/hierarchy/hierarchy_browser.jsp), selectingNCBI taxonomy, level 10 genera display list and set to include archaealsequences. Bacterial or archaeal genera not covered within the RDPIIdata were entered and parsed from a separate data file, whereappropriate. Phylogenetic distribution and grouping of genera isindicated using an ASCII based tree-abstraction. The Y-axis indicatese-value ranges, and the Z-axis (colour coded) represents the frequencyof hits for each genus in each e-value range in log-scale. RespectiveLog-colour-scales of frequencies are indicated in each figure, wherebywarmer colours indicate higher frequencies. Figure (a) allows all BLASThits per genus per ORF, accepting multiple genus hits per ORF. Figure(b) employs a frequency cutoff of one hit per genus per ORF, effectivelylimiting the hit rate to the best Blast hit found in each given ORF andgenus.

FIG. 17: Sheep antibody responses to vaccination with peptides designedagainst M. ruminantium H₄MPT methyltransferase subunits (MtrCDE) andsurface proteins and binding of antibodies to immobilised M. ruminantiumcells. (a) Vaccination with peptides designed against M1 genes. (b)Binding of antibodies to immobilised M1 cells. In the antibody-bindingexperiment a negative control (NC) serum from a sheep which had not hadcolostrum as a lamb was included, as was a sample without added serumwhich served as a blank, B.

FIG. 18-1-18-2: NRPS alignment. ClustalW (Larkin et al., 2007) alignmentof non-ribosomal peptide synthetases from M. ruminantium M1 (mru00068)and Syntrophomonas wolfei subsp. wolfei str. Goettingen (swol1094).Alignment was visualised using Jalview (Waterhouse et al., 2009).Conserved residues are shown in medium shading. Domain organisation ofM. ruminantium M1 is displayed via boxes (box marked with roundedbrackets=condensation domain; box marked with pointedbrackets=adenylation domain; box marked with triangle=phosphopantetheineattachment site; box marked with double arrow=thioester reductasedomain).

FIG. 19-1 and FIG. 19-2: Mbb. ruminantium A₁A_(o) ATP synthase PCRcloning and introduction of a hexa-histidine tag at the N-terminal ofsubunit A by PCR overlap expression. FIG. 19-1 shows (A) PCR overlapextension; (B) digested insert; and FIG. 19-2 shows (C) ligation.

FIG. 20-1 and FIG. 20-2: M. smithii A₁A_(o) ATP synthase PCR cloning andintroduction of a hexa-histidine tag by PCR overlap expression. FIG.20-1 shows (A) Cloning; FIG. 20-2 shows (B) PCR overlap extension; and(C) digested insert and ligation.

FIG. 21: (A) pTrMbrA1HIS clone which contains the genes encoding for theM. ruminantium A1-ATPase in the E. coli expression vector pTrc99a, andincludes a Hexa-Histidine tag on the N-terminal of Subunit A. (B)pTrMbrA1A0HIS9 clone.

FIG. 22: Western blot analysis of pTrMbrA1HIS expression. Lane 1:Pre-stain Protein Marker; Lane 2: Soluble Material 10 μg; Lane 3:Unbound Material 10 μg; Lane 4: Wash 1, 10 μg; Lane 5: Wash 2, 10 μg;Lane 6: Elutant 1, 10 μg (150 mM Imidazole); Lane 7: Elutant 2, 10 μg(150 mM Imidazole); Lane 8: Elutant 2, 20 μg; Lane 9: Elutant 2, 20 μg,Denatured 95° C. 5 min; Lane 10: Soluble Material 10 μg, Denatured 95°C. 5 min.

FIG. 23: Western blot analysis of pTrMbsA1HIS expression. Lane 1:Pre-stain Protein Marker; Lane 2: C41-pLysRARE/pTrMbsA1HIS startingmaterial; Lane 3: pTrMbsA1HIS unbound material; Lane 4: pTrMbsA1HIS Wash1, 40 mM imidazole; Lane 5: pTrMbsA1HIS Wash 2, 40 mM imidazole; Lane 6:pTrMbsA1HIS Elutant 1, 150 mM imidazole; Lane 7: pTrMbsA1HIS Elutant 2,150 mM imidazole; Lane 8: pTrMbsA1HIS Elutant 3, 400 mM imidazole.

FIG. 24: Growth of E. coli strains BL21 (A), DK8 (B) and C41 (C)harbouring pTRMbbr-A₁A_(O) at 37° C., showing the effect of inducedexpression of the Mbb. ruminantium A₁A_(O)-ATP synthase. (D)Localization of the Mbb. ruminantium A₁A_(O)-ATPase in DK8 by SDS-PAGEand western analysis. Samples were resolved on a 14% polyacrylamide gelin the presence of 0.1% sodium dodecyl sulfate (SDS) and either stainedwith Coomassie Brilliant blue or transferred for western analysis to apolyvinylidene difluoride membrane in the presence of 0.02% SDS and thenimmunoblotted using a penta-His antibody conjugate to detect thehexa-histidine tag on the A-subunit. Lane 1, protein molecular weightladder; Lane 2, His-tagged TA2.A1 F₁F_(o) ATPase purification (positivecontrol); Lane 3, cell lysate; Lane 4, cytoplasmic fraction; Lane 5,membrane fraction.

FIG. 25: Purification (A and B) of the Mbb. ruminantium A₁A_(O)-ATPase.

FIG. 26: Subunits of the Mbb. ruminantium A₁A_(O)-ATPase.

FIG. 27: Extraction of the A₁A_(O)-ATPase K-subunit monomer.

FIG. 28: Na⁺ binding motif in the A₁A_(O)-ATPase K-subunit.

FIG. 29-1 and FIG. 29-2: Mbb. ruminantium purified A₁A_(O) ATP synthaseactivity. (A) Kinetics of ATP hydrolysis by purified Mbb. ruminantiumA₁A_(O) ATP synthase. Background ATPase activity generated by thermalhydrolysis of ATP or contaminant ATP in buffer or enzyme has beensubtracted (these totalled <5% of the final value shown). (B) Influenceof Mg²⁺ on the kinetics of ATP hydrolysis by purified recombinant Mbb.ruminantium A₁A_(o) ATP synthase. Background ATPase activity generatedby thermal hydrolysis of ATP or contaminant ATP in buffer or enzyme hasbeen subtracted (these totalled <5% of the final value shown). (C)ATPase activity over a pH range in the presence and absence of Na⁺.Background ATPase activity generated by thermal hydrolysis of ATP orcontaminant ATP in buffer or enzyme has been subtracted from theactivities displayed (these totalled <5% of the final value shown). (D)Stability of the purified and membrane-bound (in DK8 membranes) Mbb.ruminantium A₁A_(o) ATP synthase. Background ATPase activity generatedby thermal hydrolysis of ATP or contaminant ATP in buffer or enzyme hasbeen subtracted (these totalled <5% of the final value shown).

FIG. 30-1 and FIG. 30-2: Mbb. ruminantium soluble and membrane-boundA₁A_(o) ATP synthase activity. (A and B) Effects of TBT and DCCD onATPase activity. E. coli DK8 (Δatp) inverted membranes containing therecombinant A₁A_(O)-ATPase were used to determine the effects of theinhibitors TBT (200 μM) and DCCD (250 μM) at different pH values. ATPaseactivity was measured in presence of 130 mM Na⁺ (A) and in absence ofNa⁺ (B). Background ATPase activity generated by thermal hydrolysis ofATP or contaminant ATP in buffer or enzyme has been subtracted (thesetotalled <5% of the final value shown). (C) Tributylin Inhibition of ATPHydrolysis by Purified Recombinant Mbb. ruminantium A₁A_(o) ATPsynthase. Background ATPase activity generated by thermal hydrolysis ofATP or contaminant ATP in buffer or enzyme has been subtracted (thesetotalled <5% of the final value shown). (D) Amiloride Inhibition of ATPHydrolysis of the Mbb. ruminantium A₁A_(o) ATP synthase. BackgroundATPase activity generated by thermal hydrolysis of ATP or contaminantATP in buffer or enzyme has been subtracted (these totalled <5% of thefinal value shown).

FIG. 31: Mbb. ruminantium membrane-bound A₁A_(o) ATP synthase activity.(A) Tributylin Inhibition of ATP Hydrolysis by the Mbb. ruminantiumA₁A_(o) ATP synthase in DK8 and Native Membranes. Background ATPaseactivity generated by thermal hydrolysis of ATP or contaminant ATP inbuffer or enzyme has been subtracted (these totalled <5% of the finalvalue shown). (B) Amiloride Inhibition of ATP Hydrolysis of PurifiedRecombinant Mbb. ruminantium A₁A_(o) ATP synthase in DK8 and NativeMembranes. Background ATPase activity generated by thermal hydrolysis ofATP or contaminant ATP in buffer or enzyme has been subtracted (thesetotalled <5% of the final value shown).

FIG. 32: ATP synthesis in E. coli DK8 inverted membrane vesicles. Closedsquares with no DCCD; closed triangles, a 20 min preincubation with 250μM TBT.

FIG. 33: Model of the membrane-associated sodium ion-translocatingmethyltransferase complex from methanogenic archaea.

FIG. 34: SDS-PAGE analysis of mtrF-His, mtrG-His, mtrH-His andmtrEDCBAFGH-His expression in E. coli C41 (IPTG induction, at 37° C. for5 hr).

FIG. 35: SDS-PAGE analysis of mtrA-His, mtrC-His, mtrD-His, mtrE-His,mtrH-His and mtrEDCBAFGH-His expression in E. coli C43 (IPTG induction,at 37° C. for 4 hr).

FIG. 36: Western Blot analysis of M. ruminantium surface proteinantisera against M1 cell fraction. Pre: sera obtained beforeimmunisation; Post: sera obtained after immunisation.

FIG. 37: (A) The α2β2γ2 subunit structure of MCR which contains twonickel porphinoid F430 rings and two molecules each of methylcoenzyme M(CoM) and coenzyme B (CoB). (B) The final reaction of the energyconserving pathway of methanogenic archaea in which CoM and CoB areconverted to methane and the heterodisulfide product CoM-S-S-CoB.

FIG. 38: Optical cell density was measured in pure culture over time toassess the effectiveness of potential inhibitors.

FIG. 39-1-39-3: Predicted cell surface associated adhesion-like proteinsin M1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” should be understood in the broadest possible senseand is intended to include intact monoclonal antibodies and polyclonalantibodies. It is also intended to cover fragments and derivatives ofantibodies so long as they exhibit the desired biological activity.Antibodies encompass immunoglobulin molecules and immunologically activeportions of immunoglobulin (Ig) molecules, i.e., molecules that containan antigen binding site that specifically binds (immunoreacts with) anantigen. These include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fc, Fab, Fab′, and Fab₂ fragments, and a Fabexpression library.

Antibody molecules relate to any of the classes IgG, IgM, IgA, IgE, andIgD, which differ from one another by the nature of heavy chain presentin the molecule. These include subclasses as well, such as IgG1, IgG2,and others. The light chain may be a kappa chain or a lambda chain.Reference herein to antibodies includes a reference to all classes,subclasses, and types. Also included are chimeric antibodies, forexample, monoclonal antibodies or fragments thereof that are specific tomore than one source, e.g., one or more mouse, human, or ruminantsequences. Further included are camelid antibodies or nanobodies. Itwill be understood that each reference to “antibodies” or any like term,herein includes intact antibodies, as well as any fragments,alterations, derivatives, or variants thereof.

“Altered” polynucleotides encoding peptides, polypeptides, orantibodies, as used herein, include those with deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or functionally equivalent sequence. The encodedpeptide, polypeptide, or antibody may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalentsequence. Deliberate amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity (e.g., cell association, membrane association)or immunogenic/immunological activity is retained. For example,negatively charged amino acids may include aspartic acid and glutamicacid; positively charged amino acids may include lysine and arginine;and amino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine,glycine and alanine, asparagine and glutamine, serine and threonine, andphenylalanine and tyrosine.

The amino acid molecules as noted herein, refer to oligopeptides,peptides, polypeptides, proteins or antibodies, and any fragmentsthereof, and to any naturally occurring, recombinant, synthetic, orsemi-synthetic molecules. These molecules of the invention comprise atleast 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250 aminoacids, preferably at least 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, or at least 300, 350,400, 450, or 500 amino acids. Such amino acid sequences preferablyretain the biological activity (e.g., effect on cell growth) or theimmunogenicity/immunological activity of the molecule. The amino acidmolecules noted herein are not limited to the complete, native sequenceassociated with the full-length molecule, but include also anyfragments, alterations, derivatives, and variants thereof.

“Amplification”, as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The terms “biologically active” or “functional,” as used herein, referto a peptide or polypeptide retaining one or more structural,immunogenic, or biochemical functions (e.g., cell association, membraneassociation) of a naturally occurring sequence.

The terms “cell inhibitor” or “inhibitor,” as used herein, refer toagents that decrease or block the growth or replication of microbialcells, especially methanogen cells. A cell inhibitor can act to decreaseor block, for example, cellular division. An inhibitor can decrease orblock, for example, DNA synthesis, RNA synthesis, protein synthesis, orpost-translational modifications. An inhibitor can also decrease orblock the activity of enzymes involved in the methanogenesis pathway. Aninhibitor can also target a cell for recognition by immune systemcomponents. Inhibition of a cell also includes cell killing and celldeath, for example, from lysis, apoptosis, necrosis, etc. Usefulinhibitors include, but are not limited to, anti-methanogenesiscompounds (e.g., bromoethanesulphonic acid), antibodies and antibodyfragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides,and other antibiotics as described in detail herein.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For the sequence A-G-T, thecomplementary sequence is T-C-A, the reverse complement is A-C-T and thereverse sequence is T-G-A. Complementarity between two single strandedmolecules may be partial, in which only some of the nucleic acids bind,or it may be complete when total complementarity exists between thesingle stranded molecules. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands and in the design and use of PNA molecules.

As used herein, “computer readable media” refers to any medium which canbe read and accessed directly by a computer. A “computer-based system”refers to the hardware means, software means, and data storage meansused to analyze the sequence information of the present invention.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding a peptide, polypeptide, orantibody, or a nucleic acid complementary thereto. Such modificationsinclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. In preferred aspects, a nucleic acid derivative encodes apeptide, polypeptide, or antibody which retains a biological orimmunogenicity/immunological activity of the natural molecule. Aderivative peptide, polypeptide, or antibody is one which is modified byglycosylation, pegylation, or any similar process which retains one ormore biological function (e.g., cell association, membrane association)or immunogenicity/immunological activity of the sequence from which itwas derived.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology (i.e., less than 100%identity) or complete homology (i.e., 100% identity). A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to usingthe functional term “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (e.g., Southern ornorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or hybridizationprobe will compete for and inhibit the binding of a completelyhomologous sequence to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction.

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

An “immunogenic epitope” is defined as a part of a protein that elicitsan antibody response when the whole protein is the immunogen. On theother hand, a region of a protein molecule to which an antibody can bindis defined as an “antigenic epitope.” An “insertion” or “addition”, asused herein, refers to a change in an amino acid or nucleotide sequenceresulting in the addition of one or more amino acid residues ornucleotides, respectively, as compared to the naturally occurringmolecule.

A “methanogen,” as used herein, refers to microbes that produce methanegas, which include Methanobrevibacter, Methanothermobacter,Methanomicrobium, Methanobacterium, and Methanosarcina. Specificmethanogens include, but are not limited to, Methanobrevibacterruminantium (i.e., the M1 strain (also called “M1”), or strain DSM1093hypertext transferprotocol://www.dsmz.de/microorganisms/html/strains/strain.dsm001093.htm),Methanobrevibacter smithii, Methanobrevibacter acididurans,Methanobrevibacter thaueri, Methanobacterium bryantii, Methanobacteriumformicicum, Methanothermobacter marburgensis, Methanothermobacterwolfeii, Methanosphaera stadtmanae, Methanomicrobium mobile,Methanosarcina barkeri, Methanosarcina mazei, Methanococcoides burtonii,and Methanolobus taylorii. All methanogen genera and species areencompassed by this term.

“Microbial” cells as used herein, refers to naturally-occurring orgenetically modified microbial cells including archaebacteria such asmethanogens, halophiles, and thermoacidophiles, and eubacteria, such ascyanobacteria, spirochetes, proteobacteria, as well as Gram positive andGram negative bacteria.

The term “modified” refers to altered sequences and to sequencefragments, variants, and derivatives, as described herein.

The nucleic acid molecules as noted herein refer to polynucleotides,oligonucleotides, or fragments thereof, and to DNAs or RNAs of natural,recombinant, synthetic or semi-synthetic, origin which may be single ordouble stranded, and can represent sense or antisense strands, or codingor non-coding regions, or intergenic regions. These molecules of theinvention preferably comprise at least 12, 15, 30, 45, 60, 75, 90, 105,120, 135, 150, 300, 450, 600, 750 nucleotides, preferably at least 15 to30, 30 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 300, 300 to 450,450 to 600, or 600 to 750 nucleotides, or at least 800, 850, 900, 950,1000, 1200, 1300, 1400, or 1500 nucleotides. It will be understood thata nucleic acid molecule as noted herein, will include the native, fulllength sequence, as well as any complements, fragments, alterations,derivatives, or variants, thereof.

The term “oligonucleotide” refers to a nucleic acid sequence of at least6, 8, 10, 12, 15, 18, 21, 25, 27, 30, or 36 nucleotides, or at least 12to 36 nucleotides, or at least 15 to 30 nucleotides, which can be usedin PCR amplification, sequencing, or hybridization assays. As usedherein, oligonucleotide is substantially equivalent to the terms“amplimers,” “primers,” “oligomers,” and “probes,” as commonly definedin the art.

The term “polynucleotide,” when used in the singular or plural,generally refers to any nucleic acid sequence, e.g., anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. This includes, without limitation,single and double stranded DNA, DNA including single and double strandedregions, single and double stranded RNA, and RNA including single anddouble stranded regions, hybrid molecules comprising DNA and RNA thatmay be single stranded or, more typically, double stranded or includesingle and double stranded regions. Also included are triple-strandedregions comprising RNA or DNA or both RNA and DNA. Specifically includedare mRNAs, cDNAs, and genomic DNAs, and any fragments thereof. The termincludes DNAs and RNAs that contain one or more modified bases, such astritiated bases, or unusual bases, such as inosine. The polynucleotidesof the invention can encompass coding (e.g., SEQ ID NO: 1-1718) ornon-coding sequences (e.g., SEQ ID NO: 1719-3102 or SEQ IDNO:7607-7684), or intergenic sequences (e.g., SEQ ID NO: 3103-5866), orsense or antisense sequences, or iRNAs such as siRNAs. It will beunderstood that each reference to a “polynucleotide” or like term,herein, will include the full length sequences as well as anycomplements, fragments, alterations, derivatives, or variants thereof.

A “peptide” and “polypeptide,” as used herein, refer to the isolatedpeptides or polypeptides of the invention obtained from any species,preferably microbial, from any source whether natural, synthetic,semi-synthetic, or recombinant. Specifically, a peptide or polypeptideof the invention can be obtained from methanogen cells, such asMethanobrevibacter cells, in particular, M. ruminantium, or M. smithiicells. For recombinant production, a peptide or polypeptide of theinvention can be obtained from microbial or eukaryotic cells, forexample, Escherichia, Streptomyces, Bacillus, Salmonella, yeast, insectcells such as Drosophila, animal cells such as COS and CHO cells, orplant cells. It will be understood that each reference to a “peptide” or“polypeptide,” herein, will include the full-length sequence, as well asany fragments, alterations, derivatives, or variants, thereof.

“Peptide nucleic acid” or “PNA” as used herein, refers to an antisensemolecule or anti-gene agent which comprises bases linked via a peptidebackbone.

The term “ruminant,” as used herein, refers to animals that have a rumenas a special type of digestive organ. Ruminants include, but are notlimited to, cattle, sheep, goats, buffalo, moose, antelope, caribou, anddeer.

The term “SEQ ID NO:” refers to a specifically numbered sequence asdisclosed herein. The format of “SEQ ID NO: #-#” refers to each sequencetaken individually, and any combination thereof.

The terms “stringent conditions” or “stringency,” as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. See, e.g., Sambrook, J. et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. Numerous equivalentconditions comprising either low or high stringency depend on factorssuch as the length and nature of the sequence (DNA, RNA, basecomposition), nature of the target (DNA, RNA, base composition), milieu(in solution or immobilized on a solid substrate), concentration ofsalts and other components (e.g., formamide, dextran sulfate and/orpolyethylene glycol), and temperature of the reactions (e.g., within arange from about 5° C. below the melting temperature of the probe toabout 20° C. to 25° C. below the melting temperature). One or morefactors may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term “subject” includes human and non-human animals. Non-humananimals include, but are not limited to, birds and mammals, such asruminants, and in particular, mice, rabbits, cats, dogs, pigs, sheep,goats, cows, and horses.

The terms “substantially purified” or “isolated” as used herein, referto polypeptides, peptides, or polynucleotides, that are removed fromtheir cellular, recombinant, or synthetic environment, and are at least60% free, preferably 75% free, and most preferably at least 90% free orat least 99% free from other components with which they are associatedin their environment. “Isolated” polynucleotides and polypeptides havebeen identified and separated from at least one contaminant moleculewith which they are associated in their natural state. Accordingly, itwill be understood that isolated polynucleotides and polypeptides are ina form which differs from the form or setting in which they are found innature. It will further be appreciated that “isolated” does notnecessarily reflect the exact extent (e.g., a specific percentage) towhich the sequence has been purified.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign polynucleotides into a prokaryotic or eukaryotic host cell. Themethod is selected based on the type of host cell being transformed andmay include, but is not limited to, viral infection, electroporation,heat shock, lipofection, and particle bombardment. Such “transformed”cells include stably transformed cells in which the inserted DNA iscapable of replication either as an autonomously replicating plasmid oras part of the host chromosome. They also include cells whichtransiently express the inserted DNA or RNA for limited periods of time.

“Vaccines” as used herein include all components and compositions forstimulating the immune response in a subject. Particularly useful inthis regard are subunit vaccines, including peptide vaccines, and alsovectored vaccines, nucleic acid vaccines, and edible vaccines. Vaccinescan be used to establish or strengthen an immune response to an antigen,particularly a microbial antigen. In particular aspects, vaccinescomprise antigens that evoke host-protective reactions, e.g., antibodyformation, T helper, and T cell responses. Vaccines can also compriseantibodies, for example, for passive immunization.

A “variant” of a peptide, polypeptide, or antibody, as used herein,refers to an amino acid molecule that is altered by one or more aminoacids. A variant polynucleotide is altered by one or more nucleotides. Avariant may result in “conservative” changes, wherein a substitutedamino acid has similar structural or chemical properties, e.g.,replacement of leucine with isoleucine. More rarely, a variant mayresult in “nonconservative” changes, e.g., replacement of a glycine witha tryptophan. Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunogenic/immunological activity may be foundusing computer programs well known in the art, for example, LASERGENEsoftware (DNASTAR).

The invention also encompasses nucleic acid and amino acid variantswhich retain at least one biological activity (e.g., cell association,membrane association) or immunogenicity/immunological activity. Apreferred variant is one having substantially the same or a functionallyequivalent sequence, for example, having at least 70%, at least 75%, atleast 80%, at least 85%, and more preferably at least 90%, sequenceidentity to a disclosed sequence. A most preferred variant is one havingat least 95%, at least 97%, at least 98%, or at least 99%, at least99.5%, at least 99.8%, or at least 99.9% sequence identity to a sequencedisclosed herein. The percentage identity is determined by aligning thetwo sequences to be compared as described below, determining the numberof identical residues/nucleotides in the aligned portion, dividing thatnumber by the total number of residues/nucleotides in the inventive(queried) sequence, and multiplying the result by 100. A usefulalignment program is AlignX (Vector NTI).

DESCRIPTION OF THE INVENTION

Methane is produced in the foregut of ruminants by methanogens which actas terminal reducers of carbon in the rumen system. The multi-stepmethanogenesis pathway is well elucidated, mainly from the study ofnon-rumen methanogens. However, the adaptations that allow methanogensto grow and persist in the rumen are not well understood.Methanobrevibacter ruminantium (formerly Methanobacterium ruminantium)is the so-called type species of the Methanobrevibacter genus and wasisolated from the bovine rumen (Smith, 1958). M. ruminantium is adominant methanogen worldwide which is found in ruminants fed a widevariety of diets (Janssen, 2008). As such, M. ruminantium represents animportant target for anti-methane technology.

We have embarked on a programme to sequence the genomes of culturedrepresentatives of the main rumen methanogen groups. Defining genetargets within rumen methanogens for CH₄ mitigation technologies issomewhat akin to developing a therapeutic intervention for a microbialpathogen. Therefore, our analysis of the M. ruminantium genome ispresented with an emphasis on identifying conserved methanogen surfaceproteins suitable for vaccine development via reverse vaccinologytechniques (Rappuoli, 2001) and enzyme targets susceptible to smallmolecule inhibitors through a chemogenomics approach (Caron et al.,2001).

In view of this, we have elucidated the full genome sequence of M.ruminantium and have identified all of the components of themethanogenesis pathway therein. Comparison of these gene sequences withthose from Methanobacterium thermoautotrophicum and Methanosphaerastadtmanae indicates methanogenesis gene organisation is conservedwithin the Methanobacteriales (FIGS. 1, 10, 13, and 15). The M.ruminantium genome also includes many large surface proteins which maymediate association with other rumen microbes. Based on the role of M.ruminantium in the rumen environment, the identified polynucleotides andpolypeptides can be used as a means for inhibiting methanogens and/ormethanogenesis, and to further elucidate the role of M. ruminantium inmethane formation. Particularly useful are the disclosed polynucleotidesand polypeptides identified as components involved in methanogenesis(Tables 2, 4, 5, and 9, below), as cell surface components (Tables 3, 5,6, and 9, below), as components involved in exopolysaccharidebiosynthesis (Tables 2, 4, and 9, below), as components with membranespanning domains (Tables 3 and 9, below), as components involved innon-ribosomal peptide synthesis (Tables 7 and 9, below), as well as thepolynucleotides and polypeptides for antibody production (Tables 2 and9, below). The specific M. ruminantium sequences are disclosed herewithinclude identified ORFs (Table 11), non-coding features (Table 12), andintergenic regions (Table 13).

The M1 genome was sequenced, annotated and subjected to comparativegenomic and metabolic pathway analyses. Conserved andmethanogen-specific gene sets suitable as targets for vaccinedevelopment or chemogenomic-based inhibition of rumen methanogens wereidentified. The feasibility of using a synthetic peptide-directedvaccinology approach to target epitopes of methanogen surface proteinswas demonstrated. A prophage genome was described and its lytic enzyme,endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. Apredicted stimulation of M1 growth by alcohols was demonstrated andmicroarray analyses indicated up-regulation of methanogenesis genesduring co-culture with a hydrogen (H2) producing rumen bacterium. Wealso report the discovery of non-ribosomal peptide synthetases in M.ruminantium M1, the first reported in archaeal species. The M1 genomesequence provides new insights into the lifestyle and cellular processesof this important rumen methanogen. It also defines vaccine andchemogenomic targets for broad inhibition of rumen methanogens andrepresents a significant contribution to worldwide efforts to mitigateruminant methane emissions and reduce production of anthropogenicgreenhouse gases.

Peptides, Polypeptides, and Polynucleotides

The invention encompasses peptides and polypeptides, including thosecomprising at least one of SEQ ID NO: 5867-7584, and fragments,variants, and derivatives thereof. The peptides and polypeptides of thepresent invention may be expressed and used in various assays todetermine their biological activity. The peptides and polypeptides maybe used for large-scale synthesis and isolation protocols, for example,for commercial production. Such peptides and polypeptides may be used toraise antibodies, to isolate corresponding amino acid sequences, and toquantitatively determine levels of the amino acid sequences. Thepeptides and polypeptides can be used for vaccines for targeting andinhibiting microbial cells, especially methanogen cells. The peptidesand polypeptides can also be used for preparing antibodies to inhibitthe growth or replication of such cells. The peptides and polypeptidesof the present invention may also be used as compositions, for example,pharmaceutical compositions, especially vaccine compositions. Inparticular aspects, slow-release ruminal devices can be used inconjunction with the peptides, polypeptides, antibodies, andcompositions (e.g., pharmaceutical compositions, especially vaccinecompositions) of the invention.

The peptides of the present invention comprise at least one sequenceselected from the group consisting of: (a) peptides comprising at leasta fragment of an one amino acid sequence selected from the groupconsisting of SEQ ID NO: 5867-7584, or fragments, variants, orderivatives thereof; (b) peptides comprising a functional domain of atleast one amino acid sequence selected from the group consisting of SEQID NO: 5867-7584, and fragments and variants thereof; and (c) peptidescomprising at least a specified number of contiguous residues (seeexemplary lengths hereinabove) of at least one amino acid sequenceselected from the group consisting of SEQ ID NO: 5867-7584, or variantsor derivatives thereof. In one embodiment, the invention encompasses anisolated peptide comprising the amino acid sequence of at least one ofSEQ ID NO: 5867-7584. All of these sequences are collectively referredto herein as peptides of the invention.

The polypeptides of the present invention comprise at least one sequenceselected from the group consisting of: (a) polypeptides comprising atleast one amino acid sequence selected from the group consisting of SEQID NO: 5867-7584, or fragments, variants, or derivatives thereof; (b)polypeptides comprising a functional domain of at least one amino acidsequence selected from the group consisting of SEQ ID NO: 5867-7584, andfragments and variants thereof; and (c) polypeptides comprising at leasta specified number of contiguous residues (see exemplary lengthshereinabove) of at least one amino acid sequence selected from the groupconsisting of SEQ ID NO: 5867-7584, or variants or derivatives thereof.In one embodiment, the invention encompasses an isolated polypeptidecomprising the amino acid sequence of at least one of SEQ ID NO:5867-7584. All of these sequences are collectively referred to herein aspolypeptides of the invention.

The invention also encompasses an isolated polynucleotide that encodes apeptide or polypeptide of SEQ ID NO: 5867-7584. The isolatedpolynucleotides of the present invention have utility in genome mapping,in physical mapping, and in cloning of genes of more or less relatedcell surface components. Probes designed using the polynucleotides ofthe present invention may be used to detect the presence and examine theexpression patterns of genes in any organism having sufficientlyhomologous DNA and RNA sequences in their cells, using techniques thatare well known in the art, such as slot blot techniques or microarrayanalysis. Primers designed using the polynucleotides of the presentinvention may be used for sequencing and PCR amplifications. Thepolynucleotides of the invention can be used for preparing expressionvectors and host cells for vaccines to target and inhibit microbialcells, especially methanogen cells. The invention further encompassesthe use of the polynucleotides for the production of antibodies toinhibit the growth or replication of such cells. The polynucleotides ofthe present invention may also be used as compositions, for example,pharmaceutical compositions, especially vaccine compositions. Inparticular aspects, slow-release ruminal devices can be used inconjunction with the polynucleotides, vectors, host cells, andcompositions (e.g., pharmaceutical compositions, especially vaccinecompositions) of the invention.

The polynucleotides of the present invention comprise at least onesequence selected from the group consisting of: (a) sequences comprisinga coding sequence for at least one amino acid sequence selected from thegroup consisting of SEQ ID NO: 5867-7584, or fragments or variantsthereof; (b) complements, reverse sequences, and reverse complements ofa coding sequence for at least one amino acid sequence selected from thegroup consisting of SEQ ID NO: 5867-7584, or fragments or variantsthereof; (c) open reading frames contained in the coding sequence for atleast one amino acid sequence selected from the group consisting of SEQID NO: 5867-7584, and their fragments and variants; (d) functionaldomains of a coding sequence for at least one amino acid sequenceselected from the group consisting of SEQ ID NO: 5867-7584, andfragments and variants thereof; and (e) sequences comprising at least aspecified number of contiguous residues (see exemplary lengthshereinabove) of a coding sequence for at least one amino acid sequenceselected from the group consisting of SEQ ID NO: 5867-7584, or variantsthereof; and (f) sequences comprising at least a specified number ofcontiguous nucleotides (see exemplary lengths hereinabove) of any one ofSEQ ID NO: 1-1718. Oligonucleotide probes and primers (e.g., SEQ ID NO:7586-7607) and their variants are also provided. All of thesepolynucleotides and oligonucleotide probes and primers are collectivelyreferred to herein, as polynucleotides of the invention.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding the peptides or polypeptides of the invention, some bearingminimal homology to the nucleotide sequences of any known and naturallyoccurring gene, may be produced. Thus, the invention contemplates eachand every possible variation of nucleotide sequence that could be madeby selecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to naturally occurring amino acid sequences, and allsuch variations are to be considered as being specifically disclosed.

Nucleotide sequences which encode the peptides or polypeptides, or theirfragments or variants, are preferably capable of hybridizing to thenucleotide sequence of the naturally occurring sequence underappropriately selected conditions of peptide or stringency. However, itmay be advantageous to produce nucleotide sequences encoding a peptideor polypeptide, or its fragment or derivative, possessing asubstantially different codon usage. Codons may be selected to increasethe rate at which expression of the peptide or polypeptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encodingpeptides or polypeptides and its derivatives without altering theencoded amino acid sequences include the production of RNA transcriptshaving more desirable properties, such as a greater half-life, thantranscripts produced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode the peptides or polypeptides, or their fragmentsor variants, entirely by synthetic chemistry. After production, thesynthetic sequence may be inserted into any of the many availableexpression vectors and cell systems using reagents that are well knownin the art. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding a peptide or polypeptide, or anyvariants or fragment thereof. Also encompassed by the invention arepolynucleotide sequences that are capable of hybridizing to the claimednucleotide sequences, and in particular, those shown in SEQ ID NO:1-1718, under various conditions of stringency as taught in Wahl, G. M.and S. L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.(1987; Methods Enzymol. 152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (U.S. Biochemical Corp, Cleveland, Ohio),Taq polymerase (Perkin Elmer), thermostable T7 polymerase AmershamPharmacia Biotech (Piscataway, N.J.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System marketed by Life Technologies (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200;MJ Research, Watertown, Mass.) the ABI Catalyst and 373 and 377 DNASequencers (Perkin Elmer), or the Genome Sequencer 20™ (RocheDiagnostics).

The polynucleotides encoding the peptides or polypeptides may beextended utilizing a partial nucleotide sequence and employing variousmethods known in the art to detect upstream sequences such as promotersand regulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotides or fragmentsthereof which encode peptides or polypeptides may be used in recombinantDNA molecules to direct expression of the peptides or polypeptides, orfragments or variants thereof, in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences whichencode substantially the same or a functionally equivalent amino acidsequence may be produced, and these sequences may be used to clone andexpress peptides or polypeptides. The nucleotide sequences of thepresent invention can be engineered using methods generally known in theart in order to alter amino acid-encoding sequences for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, site-directed mutagenesis may be used to insertnew restriction sites, alter glycosylation patterns, change codonpreference, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding peptides or polypeptides maybe ligated to a heterologous sequence to encode a fusion protein. Forexample, it may be useful to encode a chimeric sequence that can berecognized by a commercially available antibody. A fusion protein mayalso be engineered to contain a cleavage site located between thepeptide or polypeptide of the invention and the heterologous proteinsequence, so that the peptide or polypeptide may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding peptides or polypeptides maybe synthesized, in whole or in part, using chemical methods well knownin the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp.Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232). Alternatively, the polypeptide itself may be produced usingchemical methods to synthesize the amino acid molecule, or a fragmentthereof. For example, polypeptide synthesis can be performed usingvarious solid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204; Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer). Various fragments of peptides orpolypeptides may be chemically synthesized separately and combined usingchemical methods to produce the full length molecule.

The newly synthesized peptide or polypeptide may be isolated bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins Structures and Molecular Principles, WH Freeman and Co.,New York, N.Y.). The composition of the synthetic peptides orpolypeptides may be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure; Creighton, supra). Additionally,the amino acid sequence of the peptide or polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant molecule.

In order to express a biologically active peptides or polypeptides, thenucleotide sequences encoding the sequences or functional equivalents,may be inserted into an appropriate expression vector, i.e., a vectorwhich contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Methods which are wellknown to those skilled in the art may be used to construct expressionvectors containing sequences encoding the peptide or polypeptide andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding the peptides or polypeptides of theinvention. These include, but are not limited to, microorganisms such asbacteria transformed with recombinant phage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids);or animal cell systems. For bacteria, useful plasmids include pET,pRSET, pTrcHis2, and pBAD plasmids from Invitrogen, pET and pCDFplasmids from Novagen, and Director™ plasmids from Sigma-Aldrich. Formethanogens, useful plasmids include, but are not limited to pME2001,pMV15, and pMP1. The invention is not limited by the expression vectoror host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like maybe used. The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO, and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) may be cloned intothe vector.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the peptide or polypeptide. Forexample, when large quantities of peptide or polypeptide are needed,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding a polypeptidemay be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like.

pGEX vectors (Promega, Madison, Wis.) may also be used to expresspeptides or polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor Xa protease cleavage sites so that thecloned peptide or polypeptide of interest can be released from the GSTmoiety at will. In the yeast, Saccharomyces cerevisiae, a number ofvectors containing constitutive or inducible promoters such as alphafactor, alcohol oxidase, and PGH may be used. For reviews, see Ausubelet al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding the peptides or polypeptides of theinvention. Such signals include the ATG initiation codon and adjacentsequences. In cases where sequences encoding a peptide or polypeptide,its initiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed peptide or polypeptide in the desired fashion. Suchmodifications of the sequence include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the peptide or polypeptide may also be used to facilitatecorrect insertion, folding, and/or function. Different host cells whichhave specific cellular machinery and characteristic mechanisms forpost-translational activities are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the sequence. Specific host cellsinclude, but are not limited to, methanogen cells, such asMethanobrevibacter cells, in particular, M. ruminantium, or M. smithiicells. Host cells of interest include, for example, Rhodotorula,Aureobasidium, Saccharomyces, Sporobolomyces, Pseudomonas, Erwinia andFlavobacterium; or such other organisms as Escherichia, Lactobacillus,Bacillus, Streptomyces, and the like. Specific host cells includeEscherichia coli, which is particularly suited for use with the presentinvention, Saccharomyces cerevisiae, Bacillus thuringiensis, Bacillussubtilis, Streptomyces lividans, and the like.

There are several techniques for introducing polynucleotides intoeukaryotic cells cultured in vitro. These include chemical methods(Felgner et al., Proc. Natl. Acad. Sci., USA, 84:7413 7417 (1987);Bothwell et al., Methods for Cloning and Analysis of Eukaryotic Genes,Eds., Jones and Bartlett Publishers Inc., Boston, Mass. (1990), Ausubelet al., Short Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y. (1992); and Farhood, Annal. NY Acad. Sci., 716:23 34 (1994)),use of protoplasts (Bothwell, supra) or electrical pulses (Vatteroni etal., Mutn. Res., 291:163 169 (1993); Sabelnikov, Prog. Biophys. Mol.Biol., 62: 119 152 (1994); Bothwell et al., supra; and Ausubel et al.,supra), use of attenuated viruses (Davis et al., J. Virol. 1996, 70(6),3781 3787; Brinster et al. J. Gen. Virol. 2002, 83(Pt 2), 369 381; Moss,Dev. Biol. Stan., 82:55 63 (1994); and Bothwell et al., supra), as wellas physical methods (Fynan et al., Int J Immunopharmacol. 1995 February;17(2):79-83; Johnston et al., Meth. Cell Biol., 43(Pt A):353 365 (1994);Bothwell et al., supra; and Ausubel et al., supra).

Successful delivery of polynucleotides to animal tissue can be achievedby cationic liposomes (Watanabe et al., Mol. Reprod. Dev., 38:268 274(1994)), direct injection of naked DNA or RNA into animal muscle tissue(Robinson et al., Vacc., 11:957 960 (1993); Hoffman et al., Vacc.12:1529 1533; (1994); Xiang et al., Virol., 199:132 140 (1994); Websteret al., Vacc., 12:1495 1498 (1994); Davis et al., Vacc., 12:1503 1509(1994); Davis et al., Hum. Molec. Gen., 2:1847 1851 (1993); Dalemans etal. Ann NY Acad. Sci. 1995, 772, 255 256. Conry, et al. Cancer Res.1995, 55(7), 1397-1400), and embryos (Naito et al., Mol. Reprod. Dev.,39:153 161 (1994); and Burdon et al., Mol. Reprod. Dev., 33:436 442(1992)), intramuscular injection of self replicating RNA vaccines (Daviset al., J Virol 1996, 70(6), 3781 3787; Balasuriya et al. Vaccine 2002,20(11 12), 1609 1617) or intradermal injection of DNA using “gene gun”technology (Johnston et al., supra).

A variety of protocols for detecting and measuring the expression of thepeptides or polypeptides of the invention, using either polyclonal ormonoclonal antibodies specific for the protein are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay can be used with monoclonalantibodies reactive to two non-interfering epitopes on the peptide orpolypeptide, but a competitive binding assay can also be used. These andother assays are described, among other places, in Hampton, R. et al.(1990; Serological Methods, a laboratory Manual, APS Press, St Paul,Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences encoding the peptides orpolypeptides, or any fragments or variants thereof, may be cloned into avector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and may be used to synthesize RNAprobes in vitro by addition of an appropriate RNA polymerase such as T7,T3, or SP6 and labeled nucleotides. These procedures may be conductedusing a variety of commercially available kits Amersham PharmaciaBiotech, Promega, and US Biochemical. Suitable reporter molecules orlabels, which may be used for ease of detection, include radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Expression vectors or host cells transformed with expression vectors maybe cultured under conditions suitable for the expression and recovery ofthe peptide or polypeptide from culture. The culture can comprisecomponents for in vitro or in vivo expression. In vitro expressioncomponents include those for rabbit reticulocyte lysates, E. colilysates, and wheat germ extracts, for example, Expressway™ or RiPssystems from Invitrogen, Genelator™ systems from iNtRON Biotechnology,EcoPro™ or STP3™ systems from Novagen, TNT@ Quick Coupled systems fromPromega, and EasyXpress systems from QIAGEN. The peptide or polypeptideproduced from culture may be secreted or contained intracellularlydepending on the sequence and/or the vector used. In particular aspects,expression vectors which encode a peptide or polypeptide can be designedto contain signal sequences which direct secretion of the peptide orpolypeptide through a prokaryotic or eukaryotic cell membrane. Specificsignal peptides for use herein have been disclosed in detail in U.S.60/975,104 filed 25 Sep. 2007, and in PCT/2008/000247 filed 25 Sep.2008, which are hereby incorporated by reference herein in theirentirety.

Other constructions may include an amino acid domain which willfacilitate purification of the peptide or polypeptide. Such domainsinclude, but are not limited to, metal chelating domains such ashistidine-tryptophan (e.g., 6×-HIS) modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAG®extension/affinity purification system (Immunex Corp., Seattle, Wash.).Useful epitope tags include 3×FLAG®, HA, VSV-G, V5, HSV, GST, GFP, MBP,GAL4, and β-galactosidase. Useful plasmids include those comprising abiotin tag (e.g., PinPoint™ plasmids from Promega), calmodulin bindingprotein (e.g., pCAL plasmids from Stratagene), streptavidin bindingpeptide (e.g., InterPlay™ plasmids from Stratagene), a c-myc or FLAG®tag (e.g., Immunoprecipitation plasmids from Sigma-Aldrich), or ahistidine tag (e.g., QIAExpress plasmids from QIAGEN).

To facilitate purification, expression vectors can include cleavablelinker sequences such as those specific for Factor Xa or enterokinase(Invitrogen, San Diego, Calif.). For example, the vector can include oneor more linkers between the purification domain and the peptide orpolypeptide. One such expression vector provides for expression of afusion protein comprising a peptide or polypeptide of the invention anda polynucleotide encoding 6 histidine residues preceding a thioredoxinor an enterokinase cleavage site. The histidine residues facilitatepurification on IMAC (immobilized metal ion affinity chromatography asdescribed in Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281)while the enterokinase cleavage site provides a means for purifying thepeptide or polypeptide from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion can be an immunogenic (elicitingan immune response) or antigenic (antibody-binding) epitope. Theseimmunogenic epitopes can be confined to a few loci on the molecule. Itis understood that the number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983). Antibodies that react withpredetermined sites on proteins are described in Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Peptides that are extremelyhydrophobic and those of six or fewer residues generally are ineffectiveat inducing antibodies that bind to the mimicked protein; longer,peptides, especially those containing proline residues, usually areeffective. Sutcliffe et al., p. 661.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., p. 663. Theantibodies raised by antigenic epitope-bearing peptides or polypeptidesare useful to detect the mimicked protein, and antibodies to differentpeptides may be used for tracking the fate of various regions of aprotein precursor which undergoes post-translational processing. Thepeptides and anti-peptide antibodies may be used in a variety ofqualitative or quantitative assays for the mimicked protein, forinstance in competition assays since it has been shown that even shortpeptides (e.g., about 9 amino acids) can bind and displace the largerpeptides in immunoprecipitation assays. See, for instance, Wilson etal., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies of theinvention also are useful for purification of the mimicked protein, forinstance, by adsorption chromatography using methods well known in theart.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30 to about 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are considered epitope-bearingpeptides or polypeptides of the invention and also are useful forinducing antibodies that react with the mimicked protein. Preferably,the amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues and highly hydrophobicsequences are preferably avoided); and sequences containing prolineresidues are particularly preferred.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using polynucleotides of the invention. Forinstance, a short epitope-bearing amino acid sequence may be fused to alarger polypeptide which acts as a carrier during recombinant productionand purification, as well as during immunization to produce anti-peptideantibodies. Epitope-bearing peptides also may be synthesized using knownmethods of chemical synthesis. See, e.g., Houghten, R. A. (1985) Generalmethod for the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This process isfurther described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).In this procedure the individual resins for the solid-phase synthesis ofvarious peptides are contained in separate solvent-permeable packets,enabling the optimal use of the many identical repetitive steps involvedin solid-phase methods. A completely manual procedure allows 500-1000 ormore syntheses to be conducted simultaneously. Houghten et al., p. 5134

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen Virol. 66:2347-2354 (1985). Generally, animals may be immunized withfree peptide; however, anti-peptide antibody titer may be boosted bycoupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 g peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomer which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

As one of skill in the art will appreciate, the polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been demonstrated, e.g., for chimeric proteins consistingof the first two domains of the human CD4-polypeptide and variousdomains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric protein or proteinfragment alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)).

Non-Ribosomal Peptide Synthetases

As particular examples, the polypeptides of the invention may includenon-ribosomal peptide synthetases. Non-ribosomal peptides aresynthesized on enzymatic thiotemplates termed non-ribosomal peptidesynthetases (NRPS). The non-ribosomal peptides encompass a wide range ofcompounds having diverse activities including, but not limited to,immunosupressive (such as cyclosporin), surfactant (such as surfactin),siderophores (such as enterobactin), virulence factors (such asyersinabactin), antibacterial (such as penicillin and vancomycin), andanti-cancer (such as actinomycin and bleomycin) activities (Weber etal., Current Genomics 1994; 26:120-25; Ehmann et al., Proc. Nat. Acad.Sci. 2000; 97:2509-14; Gehring et al., Biochemistry 1998; 37:11637;Kallow et al., Biochemistry 1998; 37:5947-52; Trauger et al., Proc. Nat.Acad. Sci. 2000; 97:3112-17; Schauweker et al., J. Bacteriology 1999;27:2468-74; and Shen et al., Bioorganic Chem 1999; 27:155-71). As toparticular NRPS products, see Felnagle et al., Nonribosomal peptidesynthetases involved in the production of medically relevant naturalproducts, Mol. Pharm. 2008 March-April; 5(2):191-211.

Non-ribosomal peptides typically range in size from 1-11 amino acids andare produced by a variety of microbes including cyanobacteria,actinomycetes, and fungi. In many cases the non-ribosomal peptidescontain non-proteogenic amino acids such as norleucine, β-alanine,ornithine, etc., for which biogenesis pathways, which are secondary toprimary metabolism, are required and are post-synthetically modified(e.g., hydroxylated, methylated or acylated) by tailoring enzymes. Theterm proteogenic indicates that the amino acid can be incorporated intoa protein in a cell through well-known metabolic pathways. The choice ofincluding a (D)- or (L)-amino acid into a peptide of the presentinvention depends, in part, on the desired characteristics of thepeptide. For example, the incorporation of one or more (D)-amino acidscan confer increasing stability on the peptide in vitro or in vivo. Theterm amino acid equivalent refers to compounds which depart from thestructure of the naturally occurring amino acids, but which havesubstantially the structure of an amino acid, such that they can besubstituted within a peptide that retains biological activity. Thus, forexample, amino acid equivalents can include amino acids having sidechain modifications and/or substitutions, and also include relatedorganic acids, amides or the like.

The polynucleotide sequences required to make a NRPS and the necessarytailoring enzymes have been shown in all cases to be localized to thechromosome of the producing microbe. NRPS are modular in nature, where amodule may be defined as a segment of the NRPS necessary to catalyze theactivation of a specific amino acid and result in the incorporation ofthat amino acid into a non-ribosomal peptide. A minimal module containsthree domains: (1) adenylation domains (about 60 kDa), responsible forselecting and activating an amino acid and transferring the aminoacyladenylate to a peptidyl carrying centre; (2) thiolation domains, alsoreferred to as peptidyl carrier proteins (8-10 kDa), containing a serineresidue which is post-translationally modified with a4-phosphopantetheine group (Ppant) which acts as an acceptor for theaminoacyl adenylate; and (3) condensation domains (50-60 kDa) whichcatalyze peptide bond-forming chain-translocating steps between anupstream peptidyl-s-Ppant and the downstream aminoacyl-Ppant of theadjacent module (Doekel, S. and Marahiel, M. A. 2000; Chem. Biol.7:373-384). This minimal module for chain extension is typicallyrepeated within a synthetase and a co-linear relationship exists betweenthe number of modules present and the number of amino acids in the finalproduct with the order of the modules in the synthetase determining theorder of the amino acids in the peptide.

The adenylation domain is typically about 60 kDa. The main function ofthis domain is to select and activate a specific amino acid as anaminoacyl adenylate. Based on its function, the adenylation domainregulates the sequence of the peptide being produced. Once charged (asan amino acyl adenylate moiety), the amino acid is transferred to athiolation domain (peptidyl carrying centre). The thiolation domain isalso referred to as a peptidyl carrier protein. This domain is typically8-10 kDa and contains a serine residue that is post-translationallymodified with a 4-phosphopantetheine group. This group acts as anacceptor for the aminoacyl adenylate moiety on the amino acid. Anucleophilic reaction leads to the release of the aminoacyl adenylateand conjugation of the amino acid to thiolation domain via a thioesterbond. The condensation domain is typically about 50-60 kDa in size. Themain function of this domain is to catalyze the formation of a peptidebond between two amino acids. In this reaction an upstream tetheredpeptidyl group is translocated to the downstream aminoacyl-s-Ppant andlinked to the amino acid by peptide bond formation.

This minimal module for chain extension is typically repeated within asynthetase. Additionally, and typically, a co-linear relationship existsbetween the number of modules present and the number of amino acids inthe final product with the order of the modules in the synthetasedetermining the order of the amino acids in the peptide. This 1:1relationship, with every amino acid in the product having one modulewithin the enzyme, is referred to as the co-linearity rule. Exampleshave been found that violate this rule, and in such cases, the NRPScontains more modules than one would expect based on the number of aminoacids incorporated in the peptide product (Challis et al., Chem. Biol.2000; 7:211-24). In some cases the minimal module also is supplementedwith additional domains (epimerization, N- or C-methylation, orcyclization domain), with their position in the synthetase determiningthe substrate upon which they can act. In addition, it has been observedthat NRPS contain inter-domain spacers or linker regions. It has beenproposed that these spacers may play a critical role in communicationbetween domains, modules, and even entire synthetases.

There are highly conserved motifs in the catalytic domains of peptidesynthetases including: 10 conserved motifs in the adenylation domain; 1conserved motif in the thiolation domain; 7 conserved motifs in thecondensation domain; 1 conserved motif in the thioesterase domain; 7conserved motifs in the epimerization domains; and 3 conserved motifs inthe N-methylation domains. These are detailed in Marahiel et al.,Chemical Rev. 1997; 97:2651-73. In addition to modifications such asepimerization, methylation and cyclization during peptide synthesis,post-translational modifications including methylation, hydroxylation,oxidative cross-linking and glycosylation can occur (Walsh et al., Curr.Opin. Chem. Biol. 2001; 5:525-34).

In the present invention, the polynucleotide and polypeptide sequencesfor NRPS from M. ruminantium have been characterized (see below). Foruse with the present invention, the enzymes may be tailored as needed.For example, after production of the core of the peptide product, thesequence may then be modified by additional enzymes which are hereintermed tailoring enzymes. These enzymes alter the amino acids in thecompound without altering the number or the specific amino acids presentwithin the compound. Such tailoring enzymes may include, but are notlimited to, arginine cyclase, an O-mannosyltransferase, a phenylalanineC-methyltransferase, a first isovaleryl transferase, and a secondisovaleryl transferase. The present invention permits specific changesto be made, either by site directed mutagenesis or replacement, togenetically modify the peptide core. The modifications may be made in arational manner to improve the biological activity of the peptideproduced by the strain or to direct synthesis of compounds that arestructurally related to the peptide. The invention also allows for thetailoring enzymes to be used for biotransformation experiments toproduce enzymes to modify and possibly improve other useful compounds.As to modified NRPS products, see Velkov et al., Non-ribosomal peptidesynthetases as technological platforms for the synthesis of highlymodified peptide bioeffectors—Cyclosporin synthetase as a complexexample, Biotechnol Annu Rev. 2003; 9:151-97.

The determination of the NRPS from M. ruminantium also enables one ofordinary skill in the art to clone and express the pathway into aheterologous organism. Any organism may be used, although preferably abacterial strain is used. The choice of organism is dependent upon theneeds of the skilled artisan. For example, a strain that is amenable togenetic manipulation may be used in order to facilitate modification andproduction of the peptide product. The present invention advantageouslypermits specific changes to be made to individual modules of NRPS,either by site directed mutagenesis or replacement, to geneticallymodify the peptide core. Additionally, the NRPS modules can be used tomodify other NRPS that direct the synthesis of other useful peptidesthrough module swapping. For example, the module in NRPS thatincorporates tyrosine into the peptide core of the product may bemodified so as to incorporate a serine in its place.

The activity of any NRPS disclosed herein may be evaluated using anymethod known in the art. For example, specific modifications to thepolypeptide sequence may be produced to alter the final product. Othernon-limiting examples of studies that may be conducted with theseproteins include (i) evaluation of the biological activity of a proteinand (ii) manipulation of a synthetic pathway to alter the final productfrom microbes. Genetic manipulations and expression of the polypeptidesdiscussed herein may be conducted by any method known in the art. Forexample, the effect of point mutations may be evaluated. The mutationsmay be produced by any method known in the art. In one specific method,the manipulations and protein expression may be conducted using a vectorthat comprises at least one origin of replication. The origins ofreplication allow for replication of the polynucleotide in the vector inthe desired cells. Additionally, the vector may comprise a multiplecloning site that allows for the insertion of a heterologous nucleicacid that may be replicated and transcribed by a host cell. In oneparticular aspect, conjugation can be used for the direct transfer ofnucleic acid from one prokaryotic cell to another via direct contact ofcells. The origin of transfer is determined by a vector, so that bothdonor and recipient cells obtain copies of the vector. Transmissibilityby conjugation is controlled by a set of genes in the tra region, whichalso has the ability to mobilize the transfer of chromosomes when theorigin of transfer is integrated into them (Pansegrau et al., J. Mol.Biol., 239:623-663, 1994; Fong and Stanisich, J. Bact., 175:448-456,1993).

The vector described previously may be used to assess the biologicalactivity of the NRPS. The vector may be used to alter a polypeptide,either by partial or complete removal of the polynucleotide sequenceencoding the protein, or by disruption of that sequence. Evaluation ofthe products produced when the altered polypeptide is present is usefulin determining the functionality of the polypeptide. As discussed above,specific polypeptides within the biochemical pathway may be modified toassess the activity of the compounds produced by these alteredpolypeptides and to determine which sections of the product areimportant for activity and function. The present invention contemplatesany method of altering any of the NRPS of the present invention. Morespecifically, the invention contemplates any method that would insertamino acids, delete amino acids or replace amino acids in thepolypeptides of the invention. Additionally, a whole domain in a modulein a NRPS may be replaced. Therefore, for example, the acylation domainthat incorporates tyrosine into the final product may be replaced with adomain that incorporates serine. The modifications may be performed atthe nucleic acid level. These modifications may be performed by standardtechniques and are well known within the art. Upon production of thepolynucleotide encoding the modified polypeptide, the amino acidsequence can be expressed in a host cell. Then the host cell can becultured under conditions that permit production of a product of thealtered pathway. Once the product is isolated, the activity of theproduct may be assessed using any method known in the art. The activitycan be compared to the product of the non-modified biosynthetic pathwayand to products produced by other modifications. Correlations may bedrawn between specific alterations and activity. For example, it may bedetermined that an active residue at a specific position may increaseactivity. These types of correlations will allow one of ordinary skillto determine the most preferred product structure for specifiedactivity. As to modification techniques for NRPS, see Durfahrt et al.,Functional and structural basis for targeted modification ofnon-ribosomal peptide synthetases, Ernst Schering Res Found Workshop.2005; (51):79-106.

The present invention also contemplates a method for using anintergeneric vector to manipulate, modify, or isolate a protein involvedin the synthesis of a specific product. For example, the vector of thepresent invention may be used to alter an enzyme which is involved inincorporation of an alanine residue into a peptide, so that a tyrosineresidue is incorporated instead. The effect of this modification onpeptide function may be then be evaluated for biological efficacy. Inthe above example, modifications to the enzyme may include, but are notlimited to, removal of amino acids and/or sequences that specificallyrecognize alanine and/or incorporation of amino acids and/or sequencesthat specifically recognize tyrosine. Therefore, in general terms, thevector of the present invention may be used to alter a gene sequence byinsertion of nucleic acid sequences, deletion of nucleic acid sequences,or alteration of specific bases within a nucleic acid sequence to alterthe sequence of a polypeptide of interest; thereby producing a modifiedprotein of interest. Preferably, the polypeptide of interest is involvedin the synthesis of a compound of interest. The method of modifying aprotein may comprise (i) transfecting a first microbial cell with thevector of the present invention, (ii) culturing the first microbial cellunder conditions that allow for replication of the vector, (iii)conjugating the first microbial cell with a second microbial cell underconditions that allow for the direct transfer of the vector from thefirst microbial cell to the second microbial cell, and (iv) isolatingthe second microbial cell transformed with the vector. Other method ofvector transfer are also contemplated and disclosed herein.

Antibodies and Vaccines

The antibodies of the invention may be produced using methods which aregenerally known in the art. In particular, purified peptides,polypeptides, or polynucleotides may be used to produce antibodies inaccordance with known methods. Such antibodies may include, but are notlimited to, polyclonal, monoclonal, chimeric, and single chainantibodies, Fab fragments, and fragments produced by a Fab expressionlibrary. Neutralizing antibodies, (i.e., those which inhibit function)are especially preferred for use with vaccines.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith a peptide, polypeptide, polynucleotide, or any fragment thereofwhich has immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such asaluminium hydroxide, and surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are especiallypreferable.

It is preferred that the peptides, polypeptides, or fragments used toinduce antibodies have an amino acid sequence comprising at least fiveamino acids and more preferably at least 10 amino acids. It is alsopreferable that they are identical to a portion of the amino acidsequence of the natural protein, and they may contain the entire aminoacid sequence of a small, naturally occurring molecule. Short stretchesof amino acids may be fused with those of another protein such askeyhole limpet hemocyanin and antibody produced against the chimericmolecule.

Monoclonal antibodies may be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma technique,the human B-cell hybridoma technique, and the EBV-hybridoma technique(Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985)J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of “chimericantibodies”, e.g., the combining of mouse antibody genes and humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to producespecific single chain antibodies. Antibodies with related specificity,but of distinct idiotypic composition, may be generated by chainshuffling from random combinatorial immunoglobin libraries (Burton D. R.(1991) Proc. Natl. Acad. Sci. 88:11120-3).

Those of skill in the art to which the invention relates will appreciatethe terms “diabodies” and “triabodies”. These are molecules whichcomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) by a short peptide linker that is too short toallow pairing between the two domains on the same chain. This promotespairing with the complementary domains of one or more other chains andencourages the formation of dimeric or trimeric molecules with two ormore functional antigen binding sites. The resulting antibody moleculesmay be monospecific or multispecific (e.g., bispecific in the case ofdiabodies). Such antibody molecules may be created from two or moreantibodies using methodology standard in the art to which the inventionrelates; for example, as described by Todorovska et al. (Design andapplication of diabodies, triabodies and tetrabodies for cancertargeting. J. Immunol. Methods. 2001 Feb. 1; 248(1-2):47-66).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites may also begenerated. For example, such fragments include, but are not limited to,the F(ab′)₂ fragments which can be produced by pepsin digestion of theantibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving binding specificity. Numerous protocols for competitive bindingor immunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the measurement of complex formationbetween a peptide, polypeptide, or polynucleotide and its specificantibody. A two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes is preferred, but acompetitive binding assay may also be employed (Maddox, supra).

The antibodies described herein have the ability to target and/orinhibit cells and are also useful as carrier molecules for the deliveryof additional inhibitory molecules into microbial cells. The chemistryfor coupling compounds to amino acids is well developed and a number ofdifferent molecule types could be linked to the antibodies. The mostcommon coupling methods rely on the presence of free amino (alpha-aminoor Lys), sufhydryl (Cys), or carboxylic acid groups (Asp, Glu, oralpha-carboxyl). Coupling methods can be used to link the antibody tothe cell inhibitor via the carboxy- or amino-terminal residue. In somecases, a sequence includes multiple residues that may react with thechosen chemistry. This can be used to produce multimers, comprising morethan one cell inhibitor. Alternatively, the antibody can be shortened orchosen so that reactive residues are localized at either the amino orthe carboxyl terminus of the sequence.

For example, a reporter molecule such as fluorescein can be specificallyincorporated at a lysine residue (Ono et al., 1997) usingN-α-Fmoc-Nε-1-(4,4-dimethyl-2,6dioxocyclohex-1-ylidene-3-methylbutyl)-L-lysine during polypeptidesynthesis. Following synthesis, 5- and 6-carboxyfluorescein succinimidylesters can be coupled after 4,4-dimethyl-2,6 dioxocyclohex-1-ylidene isremoved by treatment with hydrazine. Therefore coupling of an inhibitorymolecule to the antibody can be accomplished by inclusion of a lysineresidue to the polypeptide sequence, then reaction with a suitablyderivatised cell inhibitor.

EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) orthe carbodiimide coupling method can also be used. Carbodiimides canactivate the side chain carboxylic groups of aspartic and glutamic acidas well as the carboxyl-terminal group to make them reactive sites forcoupling with primary amines. The activated antibody is mixed with thecell inhibitor to produce the final conjugate. If the cell inhibitor isactivated first, the EDC method will couple the cell inhibitor throughthe N-terminal alpha amine and possibly through the amine in theside-chain of Lys, if present in the sequence.

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is aheterobifunctional reagent that can be used to link an antibody to cellinhibitors via cysteines. The coupling takes place with the thiol groupof cysteine residues. If the chosen sequence does not contain Cys it iscommon to place a Cys residue at the N- or C-terminus to obtain highlycontrolled linking of the antibody to the cell inhibitor. For synthesispurposes, it may be helpful for the cysteine to be placed at theN-terminus of the antibody. MBS is particularly suited for use with thepresent invention.

Glutaraldehyde can be used as a bifunctional coupling reagent that linkstwo compounds through their amino groups. Glutaraldehyde provides ahighly flexible spacer between the antibody and cell inhibitor forfavorable presentation. Glutaraldehyde is a very reactive compound andwill react with Cys, Tyr, and His to a limited extent. Theglutaraldehyde coupling method is particularly useful when a polypeptidecontains only a single free amino group at its amino terminus. If theantibody contains more than one free amino group, large multimericcomplexes can be formed.

In one aspect, the antibodies of the invention can be fused (e.g., byin-frame cloning) or linked (e.g., by chemical coupling) to cellinhibitors such as antimicrobial agents. Included among these areantimicrobial peptides, for example,bactericidal/permeability-increasing protein, cationic antimicrobialproteins, lysozymes, lactoferrins, and cathelicidins (e.g., fromneutrophils; see, e.g., Hancock and Chapple, 1999, Antimicrob. AgentsChemother.43:1317-1323; Ganz and Lehrer, 1997, Curr. Opin. Hematol.4:53-58; Hancock et al., 1995, Adv. Microb. Physiol. 37:135-175).Antimicrobial peptides further include defensins (e.g., from epithelialcells or neutrophils) and platelet microbiocidal proteins (see, e.g.,Hancock and Chapple, 1999, Antimicrob. Agents Chemother.43:1317-1323).Additional antimicrobial peptides include, but are not limited to,gramicidin S, bacitracin, polymyxin B, tachyplesin, bactenecin (e.g.,cattle bactenecin), ranalexin, cecropin A, indolicidin (e.g., cattleindolicidin), and nisin (e.g., bacterial nisin).

Also included as antimicrobial agents are ionophores, which facilitatetransmission of an ion, (such as sodium), across a lipid barrier such asa cell membrane. Two ionophore compounds particularly suited to thisinvention are the RUMENSIN™ (Eli Lilly) and Lasalocid (Hoffman LaRoche).Other ionophores include, but are not limited to, salinomycin,avoparcin, aridcin, and actaplanin. Other antimicrobial agents includeMonensin™ and azithromycin, metronidazole, streptomycin, kanamycin, andpenicillin, as well as, generally, B-lactams, aminoglycosides,macrolides, chloramphenicol, novobiocin, rifampin, and fluoroquinolones(see, e.g., Horn et al., 2003, Applied Environ. Microbiol. 69:74-83;Eckburg et al., 2003, Infection Immunity 71:591-596; Gijzen et al.,1991, Applied Environ. Microbiol. 57:1630-1634; Bonelo et al., 1984,FEMS Microbiol. Lett. 21:341-345; Huser et al., 1982, Arch. Microbiol.132:1-9; Hilpert et al., 1981, Zentbl. Bakteriol. Mikrobiol. Hyg. 1 AbtOrig. C 2:21-31).

Particularly useful inhibitors are compounds that block or interferewith methanogenesis, including bromoethanesulphonic acid, e.g.,2-bromoethanesulphonic acid (BES) or a salt thereof, for example, asodium salt. Sodium molybdate (Mo) is an inhibitor of sulfate reduction,and can be used with bromoethanesulphonic acid. Otheranti-methanogenesis compounds include, but are not limited to, nitrate,formate, methyl fluoride, chloroform, chloral hydrate, sodium sulphite,ethylene and unsaturated hydrocarbons, acetylene, fatty acids such aslinoleic and cis-oleic acid, saturated fatty acids such as behenic andstearic acid, and, also lumazine (e.g., 2,4-pteridinedione). Additionalcompounds include 3-bromopropanesulphonate (BPS), propynoic acid, andethyl 2-butynoate.

Further included as antimicrobial agents are lytic enzymes, includingphage lysozyme, endolysin, lysozyme, lysin, phage lysin, muralysin,muramidase, and virolysin. Useful enzymes exhibit the ability tohydrolyse specific bonds in the bacterial cell wall. Particular lyticenzymes include, but are not limited to, glucosaminidases, whichhydrolyse the glycosidic bonds between the amino sugars (e.g.,N-acetylmuramic acid and N-acetylglucosamine) of the peptidoglycan,amidases, which cleave the N-acetylmuramoyl-L-alanine amide linkagebetween the glycan strand and the cross-linking peptide, andendopeptidases, which hydrolyse the interpeptide linkage (e.g., cysteineendopeptidases) and endoisopeptidases that attack pseudomurein ofmethanogens from the family Methanobacteriaceae.

Additionally, PNAs are included as antimicrobial agents. PNAs arepeptide-nucleic acid hybrids in which the phosphate backbone has beenreplaced by an achiral and neutral backbone made fromN-(2-aminoethyl)-glycine units (see, e.g., Eurekah BioscienceCollection. PNA and Oligonucleotide Inhibitors of Human Telomerase. G.Gavory and S. Balasubramanian, Landes Bioscience, 2003). The bases A, G,T, C are attached to the amino nitrogen on the backbone viamethylenecarbonyl linkages (P. E. Nielsen et al., Science 1991. 254:1497-1500; M. Egholm et al., Nature 1993. 365: 566-568). PNAs bindcomplementary sequences with high specificity, and higher affinityrelative to analogous DNA or RNA (M. Egholm et al., supra). PNA/DNA orPNA/RNA hybrids also exhibit higher thermal stability compared to thecorresponding DNA/DNA or DNA/RNA duplexes (M. Egholm et al., supra).PNAs also possess high chemical and biological stability, due to theunnatural amide backbone that is not recognized by nucleases orproteases (V. Demidov et al., Biochem Pharmacol 1994. 48: 1310-1313).Typically, PNAs are at least 5 bases in length, and include a terminallysine. PNAs may be pegylated to further extend their lifespan (Nielsen,P. E. et al. (1993) Anticancer Drug Des. 8:53-63).

In one particular aspect, the antibodies of the invention can be fusedor linked to other antibodies or fragments thereof. The added antibodiesor antibody fragments can be directed to microbial cells, orparticularly methanogen cells, or one or more cell components. Forexample, cell surface proteins, e.g., extracellular receptors, can betargeted. In certain aspects, the antibodies or antibody fragments canbe engineered with sequences that are specifically expressed insubjects, for example, human or ruminant sequences. Also included arechimeric antibodies, for example, monoclonal antibodies or fragmentsthereof that are specific to more than one source, e.g., one or moremouse, human, or ruminant sequences. Further included are camelidantibodies or nanobodies.

The antibodies of the invention find particular use in targeting amicrobial cell, in particular, a methanogen cell. In certain aspects,the antibodies can be used to associate with or bind to the cell wall ormembrane and/or inhibit growth or replication of the cell. As such, theantibodies can be used for transient or extended attachment to the cell,or to mediate sequestration or engulfment of the cell, and/or lysis. Toeffect targeting, the microbial cell can be contacted with an antibodyas isolated from a host organism, or produced by expression vectorsand/or host cells, or synthetic or semi-synthetic chemistry as describedin detail herein. Alternately, the antibodies can be produced by thehost organism itself in response to the administration or the peptides,polypeptides, or polynucleotides disclosed herein. It is understood thatthe antibodies of the invention, as well as the correspondingpolynucleotides, expression vectors, host cells, peptides, andpolypeptides, can be used to target various microbes, for example,Methanobrevibacter ruminantium, which is the primary methanogen inruminants, and Methanobrevibacter smithii, which is the primarymethanogen in humans. In particular aspects, the antibodies, orcorresponding polynucleotides, expression vectors, host cells, peptides,or polypeptides, are delivered to subjects as a composition described indetail herein, for example, through use of a slow-release ruminaldevice.

In various aspects, the agents of the invention (e.g., one or morepeptides, polypeptides, polynucleotides, and antibodies) can be includedin a composition, for example, a pharmaceutical composition, andespecially a vaccine composition. The composition comprises, forexample: a) an isolated peptide or alteration, fragment, variant, orderivative thereof; b) an isolated polypeptide, or an alteration,fragment, variant, or derivative thereof; c) an isolated polynucleotide,or an alteration, fragment, variant, or derivative thereof; d) anexpression vector comprising this polynucleotide; e) a host cellcomprising this expression vector; or (f) an antibody, or an alteration,fragment, variant, or derivative thereof. The compositions of theinvention can be specifically packaged as part of kits for targeting,and/or inhibiting microbial cells, especially methanogen cells, inaccordance with the disclosed methods. The kits comprise at least onecomposition as set out herein and instructions for use in targetingcells or inhibiting cell growth or replication, for methanogens or othermicrobes.

For vaccines, a number of approaches can be used to increase antigenimmunogenicity, for example, by use of antigen particles; antigenpolymers and polymerization; emulsifying agents; microencapsulation ofantigens; killed bacteria and bacterial products; chemical adjuvants andcytokines; and agents for targeting antigens to antigen presenting cells(reviewed in Paul, Fundamental Immunology, 1999, Lippincott-RavenPublishers, New York, N.Y., p. 1392-1405).

To render antigens particulate, alum precipitation can be used. With theuse of aluminium hydroxide or aluminium phosphate, the antigen inquestion becomes incorporated into an insoluble, gel-like precipitate orelse is bound to preformed gel by electrostatic interactions. Antigenscan be subjected to mild heat aggregation. Antigens exhibitingself-assembly can also be used. Liposomes, virosomes, and immunostainingcomplexes (ISCOMs) are also useful for forming particulates.

To promote polymerization, nonionic block copolymers can be used asadditives to adjuvants, e.g., polymers or polyoxypropylene andpolyoxyethylene, with which antigen can be associated. These are foundas components of complex adjuvant formulations by both Syntex (SAF-1,Syntex Adjuvant Formulation-1) and Ribi Chemical Co. Carbohydratepolymers of mannose (e.g., mannan) or of β1-3 glucose (e.g., glucan) canbe used in similar fashion (Okawa Y, Howard C R, Steward M W. Productionof anti-peptide antibody in mice following immunization of mice withpeptides conjugated to mannan. J Immunol Methods 1992; 142:127-131; OhtaM, Kido N, Hasegawa T, et al. Contribution of the mannan side chains tothe adjuvant action of lipopolysaccharides. Immunology 1987;60:503-507).

Various agents can be used for emulsification, including water-in-oilemulsions, such as Freund's adjuvants (e.g., Freund's incompleteadjuvant), or other mixtures comprising tiny droplets of waterstabilized by a surfactant such as mannide monooleate in a continuousphase of mineral oil or other oils, such as squalane. An alternativeapproach is to use oil-in-water emulsions, such as MF5963 (Chiron), orother mixtures comprising oil droplets of squalene and a mixture ofemulsifying agents TWEEN80 and SPAN85, and chemical immunomodulatorssuch as derivatives or muramyl dipeptide, e.g., muramyltripeptide-phosphatidyl ethanolamine (MTP-PE) (Valensi J-P M, Carlson JR, Van Nest G A. Systemic cytokine profiles in Balb/c mice immunizedwith trivalent influenza vaccine containing MF59 oil emulsion and otheradvanced adjuvants. J Immunol 1994; 153:4029-4039). Small amounts ofpolysorbate 80 and sorbitan trioleate can also be used in the mixtures.As another example, SAF-165 (Syntex) can be used, or other oil-in-watermixtures comprising Pluronic L121, squalene, and TWEEN80.

Microcapsules, in particular, biodegradable microcapsules, can be usedto prepare controlled-release vaccines (Chang TMS. Biodegradable,semi-permeable microcapsules containing enzymes hormones, vaccines andother biologicals. J Bioeng 1976; 1:25-32; Langer R. Polymers for thesustained release of macromolecules: their use in a single step methodof immunization. Methods Enzymol 1981; 73:57-75). Cyanoacrylates areanother form of biodegradable polymer. For example,poly(butyl-2-cyanoacrylate) can be used as an adjuvant for oralimmunization (O'Hagan D T, Palin K J, Davis S S. Poly(butyl-2-cyanoacrylate) particles as adjuvants for oral immunization.Vaccine 1989; 7:213-216). Microcapsules are useful for the mucosaladministration of vaccines. Particles of very small size (nanoparticles)are particularly suitable. Digestion in the stomach can be countered byenteric coated polymers, and coating with substances that increaseintestinal absorption, as needed.

Various bacteria, other than killed M. tuberculosis, can be used asadjuvants. Where the killed bacterial preparation is itself highlyantigenic, the adjuvant properties extend to the co-administeredantigen. Useful organisms include Bordetella pertussis, Corynebacteriumparvum, and Nippostrongylus brasiliensis. Peptide and lipid componentsof bacteria can also be used. Exemplary components includeacetylmuramyl-L-alanyl-D-isoglutamine, or muramyl dipeptide (MDP)(Ellouz F, Adam A, Ciorbaru R, Lederer E. Minimal structuralrequirements for adjuvant activity of bacterial peptidoglycans. BiochemBiophys Res Commun 1974; 59:1317-1325), MDP (murabutide) (Chedid L,Parant M A, Audibert F M, et al. Biological activity of a new syntheticmuramyl dipeptide devoid of pyrogenicity. Infect Immun 1982;35:417-424), threonyl MDP (Allison A C, Byars N E. An adjuvantformulation that selectively elicits the formation of antibodies ofprotective isotypes and cell-mediated immunity. J Immunol Methods 1986;95:157-168), and MTP-P E. Lipid adjuvants can comprise LPS endotoxins ofgram-negative bacteria, such as Escherichia, Salmonella, andPseudomonas. In certain approaches, the lipid A structure can bechemically modified to lower toxicity but retain adjuvanticity, e.g., asfor monophosphoryl lipid A (MPL) (Johnson A G, Tomai M, Solem L, Beck L,Ribi E. Characterization of non-toxic monophosphoryl lipid. Rev InfectDis 1987; 9:S512).

Various chemicals can be used as adjuvants, including polynucleotides,such as poly-I:C and poly-A:U, vitamin D3, dextran sulphate, inulin,dimethyl dioctadecyl ammonium bromide (DDA), avridine, carbohydratepolymers similar to mannan, and trehalose dimycolate (Morein B,Lovgren-Bengtsson K, Cox J. Modern adjuvants: functional aspects. In:Kaufmann SHE, ed. Concepts in vaccine development. Berlin: Walter deGruyter, 1996:243-263). Also included are polyphosphazines (initiallyintroduced as slow release-promoting agents) and a Leishmania protein,LeIF. Cytokines can also be used as adjuvants, for example, IL-2, IL-4,IL-6, IL-10, GM-CSF, and IFN-g.

For targeting antigen presenting cells, C3d domains, Fc domains, and CTBdomains can be used (Dempsey P W, Allison M E D, Akkaraju S, Goodnow CC, Fearon D T. C3d of complement as a molecular adjuvant: bridginginnate and acquired immunity. Science 1996; 271:348-350; Sun J-B,Holmgren J, Czerkinsky C. Cholera toxin B subunit: an efficienttransmucosal carrier-delivery system for induction of peripheralimmunological tolerance. Proc Natl Acad Sci USA 1994; 91:10795-10799;Sun J-B, Rask C, Olsson T, Holmgren J, Czerkinsky C. Treatment ofexperimental autoimmune encephalomyelitis by feeding myelin basicprotein conjugated to cholera toxin B subunit. Proc Natl Acad Sci USA1996; 93:7196-7201).

Specific adjuvants for mucosal delivery, e.g., CT, LT, and Fragment C oftetanus toxin, can also be used (Elson C J, Ealding W. Generalizedsystemic and mucosal immunity in mice after mucosal stimulation withcholera toxin. J Immunol 1984; 132:2736-2743; Holmgren J, Lycke N,Czerkinsky C. Cholera toxin and cholera B subunit as oral-mucosaladjuvant and antigen vector systems. Vaccine 1993; 11:1179-1184;Clements J D, Hartzog N M, Lyon F L. Adjuvant activity of Escherichiacoli heat-labile enterotoxin and effect on the induction of oraltolerance in mice to unrelated protein antigens. Vaccine 1988;6:269-277; Gomez-Duarte O G, Galen J, Chatfield S N, Rappuoli R, EidelsL, Levine M M. Expression of fragment C of tetanus toxin fused to acarboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD908 vaccine strain. Vaccine 1995; 13:1596-1602).

Therapeutics and Diagnostics

The peptides, polypeptides, polynucleotides, and antibodies of thepresent invention are considered to have health benefits. In particularaspects, vaccines that target methanogens can be used to restore energyto the subject that is normally lost as methane. The invention thereforerelates to a pharmaceutical composition (especially a vaccinecomposition) in conjunction with a pharmaceutically acceptable carrier,for use with any of the methods discussed above. Such pharmaceuticalcompositions may comprise a peptide, polypeptide, or antibody incombination with a cell inhibitor. Alternatively, the pharmaceuticalcompositions may comprise a polynucleotide, expression vector, or hostcell as described in detail herein. The compositions may be administeredalone or in combination with at least one other agent, such asstabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a subject alone, or in combination with other agents,drugs (e.g., antimicrobial drugs), or hormones.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.). The pharmaceutical compositions utilizedin this invention may be administered by any number of routes including,but not limited to, oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the subject. Pharmaceutical preparations for oral usecan be obtained through combination of active compounds with solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are carbohydrate orprotein fillers, such as sugars, including lactose, sucrose, mannitol,or sorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilising agents may be added, such as the crosslinked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. For topical or nasal administration, penetrantsappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation may be alyophilized powder which may contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use. After pharmaceuticalcompositions have been prepared, they can be placed in an appropriatecontainer and labeled for treatment of an indicated condition. Foradministration of a composition of the invention, such labeling wouldinclude amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For any compound, thetherapeutically effective dose can be estimated initially either in cellassays, e.g., in microbial cells, or in particular, in methanogen cells,or in animal models, usually mice, rabbits, dogs, or pigs, or inruminant species such as sheep, cattle, deer, and goats. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration. Normal dosageamounts may vary from 0.1 to 100,000 micrograms, up to a total dose ofabout 1 g, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in the literatureand generally available to practitioners in the art. Those skilled inthe art will employ different formulations for polynucleotides than forpolypeptides. Similarly, delivery of peptides, or polypeptides,polynucleotides, or antibodies will be specific to particular cells,conditions, locations, etc.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeagent or to maintain the desired effect. Factors which may be taken intoaccount include the severity of the disease state, general health of thesubject, age, weight, and gender, diet, time, and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Long-acting pharmaceutical compositionsmay be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularformulation. The compositions can be co-administered with one or moreadditional anti-microbial agents, including anti-methanogenesiscompounds (e.g., bromoethanesulphonic acid), antibodies and antibodyfragments, lytic enzymes, peptide nucleic acids, antimicrobial peptides,and other antibiotics as described in detail herein. Co-administrationcan be simultaneous or sequential, or can alternate with repeatedadministration.

Particularly useful for the compositions of the invention (e.g.,pharmaceutical compositions) are slow release formulas or mechanisms.For example, intra-ruminal devices include, but are not limited to, TimeCapsule™ Bolus range by Agri-Feeds Ltd., New Zealand, originallydeveloped within AgResearch Ltd., New Zealand, as disclosed in WO95/19763 and NZ 278977, and CAPTEC by Nufarm Health & Sciences, adivision of Nufarm Ltd., Auckland, New Zealand, as disclosed in AU35908178, PCT/AU81/100082, and Laby et al., 1984, Can. J. Anim. Sci. 64(Suppl.), 337-8, all of which are incorporated by reference herein. As aparticular example, the device can include a spring and plunger whichforce the composition against a hole in the end of a barrel.

As a further embodiment, the invention relates to a composition for awater supplement, e.g., drenching composition, or food supplement, e.g.,ruminant feed component, for use with any of the methods discussedabove. In particular aspects, the food supplement comprises at least onevegetable material that is edible, and a peptide or polypeptide of theinvention. Alternatively, the food supplement comprises at least onevegetable material that is edible, and a polypeptide or peptide, or apolynucleotide encoding a peptide or polypeptide disclosed herein, forexample, as an expression vector or host cell comprising the expressionvector. In particular, the composition further includes a cellinhibitor, as fused or linked to the resultant sequence. The preferredvegetable material include any one of hay, grass, grain, or meal, forexample, legume hay, grass hay, corn silage, grass silage, legumesilage, corn grain, oats, barley, distillers grain, brewers grain, soybean meal, and cotton seed meal. In particular, grass silage is usefulas a food composition for ruminants. The plant material can begenetically modified to contain one or more components of the invention,e.g., one or more polypeptides or peptides, polynucleotides, or vectors.

In another embodiment, antibodies which specifically bind the peptides,polypeptides, or polynucleotides of the invention may be used todetermine the presence of microbes, especially methanogens, or in assaysto monitor levels of such microbes. The antibodies useful for diagnosticpurposes may be prepared in the same manner as those described above.Diagnostic assays include methods which utilize the antibody and a labelto detect a peptide or polypeptide in human body fluids or extracts ofcells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by joining them, either covalently ornon-covalently, with a reporter molecule. A wide variety of reportermolecules which are known in the art may be used, several of which aredescribed above.

A variety of protocols for measuring levels of a peptide, polypeptide,or polynucleotide are known in the art (e.g., ELISA, RIA, and FACS), andprovide a basis for diagnosing the presence or levels of a microbe,especially a methanogen. Normal or standard levels established bycombining body fluids or cell extracts taken from normal subjects, e.g.,normal humans or ruminants, with the antibody under conditions suitablefor complex formation. The amount of standard complex formation may bequantified by various methods, but preferably by photometric means.Quantities of peptide, polypeptide, or polynucleotide expressed insubject, control, and treated samples (e.g., samples from vaccinatedsubjects) are compared with the standard values. Deviation betweenstandard and subject values establishes the parameters for determiningthe presence or levels of the microbe.

In another embodiment of the invention, the polynucleotides may be usedfor diagnostic purposes using particular hybridization and/oramplification techniques. The polynucleotides which may be used includeoligonucleotides, complementary RNA and DNA molecules, and PNAs. Thepolynucleotides may be used to detect and quantitate gene expression insamples in which expression may be correlated with the presence orlevels of a microbe. The diagnostic assay may be used to distinguishbetween the absence, presence, and alteration of microbe levels, and tomonitor levels during therapeutic intervention.

In one aspect, hybridization with PCR probes may be used to identifynucleic acid sequences, especially genomic sequences, which encode thepeptides or polypeptides of the invention. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., in the 3′ coding region, and the stringency of the hybridizationor amplification (maximal, high, intermediate, or low) will determinewhether the probe identifies only naturally occurring sequences,alleles, or related sequences. Probes may also be used for the detectionof related sequences, and should preferably contain at least 50% of thenucleotides from any of the coding sequences. The hybridization probesof the subject invention may be DNA or RNA and derived from thenucleotide sequence of SEQ ID NO: 1-1718, or complements, or modifiedsequences thereof, or from genomic sequences including promoter andenhancer elements of the naturally occurring sequence.

Means for producing specific hybridization probes for DNAs include thecloning of polynucleotides into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as ³²P or ³⁵S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like. The polynucleotides may beused in Southern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dipstick, pin, ELISA assays, ormicroarrays utilizing fluids or tissues from subject biopsies to detectthe presence or levels of a microbe. Such qualitative or quantitativemethods are well known in the art.

In a particular aspect, the polynucleotides may be useful in variousassays labelled by standard methods, and added to a fluid or tissuesample from a subject under conditions suitable for hybridization and/oramplification. After a suitable incubation period, the sample is washedand the signal is quantitated and compared with a standard value. If theamount of signal in the test sample is significantly altered from thatof a comparable control sample, the presence of altered levels ofnucleotide sequences in the sample indicates the presence or levels ofthe microbe. Such assays may also be used to evaluate the efficacy of aparticular vaccination regimen in animal studies, in clinical trials, orin monitoring the treatment of a subject.

In order to provide a basis for the diagnosis of the presence or levelsof a microbe, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, with a polynucleotide or a fragmentthereof, under conditions suitable for hybridization and/oramplification. Standard levels may be quantified by comparing the valuesobtained from normal subjects with those from an experiment where aknown amount of a substantially purified polynucleotide is used.Standard values obtained from normal samples may be compared with valuesobtained from samples from subjects treated for microbial growth.Deviation between standard and subject values is used to establish thepresence or levels of the microbe.

Once the microbe is identified and a vaccination protocol is initiated,hybridization and/or amplification assays may be repeated on a regularbasis to evaluate whether the level of expression in the subject beginsto decrease relative to that which is observed in the normal subject.The results obtained from successive assays may be used to show theefficacy of vaccination over a period ranging from several days tomonths.

Particular diagnostic uses for oligonucleotides designed from thepolynucleotides may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced in vitro.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5′.fwdarw.3′) and another with antisense orientation(3′.fwdarw.5′), employed under optimized conditions for identificationof a specific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate expression includeradiolabeling or biotinylating nucleotides, coamplification of a controlnucleic acid, and standard curves onto which the experimental resultsare interpolated (Melby, P. C. et al. (1993) J. Immunol. Methods,159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The speedof quantitation of multiple samples may be accelerated by running theassay in an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotides described herein may be used as targetsin a microarray. The microarray can be used to monitor the expressionlevel of large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of disease, to diagnose disease, and to develop andmonitor the activities of therapeutic agents. In one embodiment, themicroarray is prepared and used according to methods known in the artsuch as those described in PCT application WO 95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619).

In one aspect, the oligonucleotides may be synthesized on the surface ofthe microarray using a chemical coupling procedure and an ink jetapplication apparatus, such as that described in PCT application WO95/251116 (Baldeschweiler et al.). In another aspect, a “gridded” arrayanalogous to a dot or slot blot (HYBRIDOT apparatus, Life Technologies)may be used to arrange and link cDNA fragments or oligonucleotides tothe surface of a substrate using a vacuum system, thermal, UV,mechanical or chemical bonding procedures. In yet another aspect, anarray may be produced by hand or by using available devices, materials,and machines (including multichannel pipettors or robotic instruments;Brinkmann, Westbury, N.Y.) and may include, for example, 24, 48, 96,384, 1024, 1536, or 6144 spots or wells (e.g., as a multiwell plate), ormore, or any other multiple from 2 to 1,000,000 which lends itself tothe efficient use of commercially available instrumentation.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a biological sample. The biologicalsamples may be obtained from any bodily fluid (blood, urine, saliva,phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissuepreparations. To produce probes, the polynucleotides extracted from thesample are used to produce polynucleotides which are complementary tothe nucleic acids on the microarray. If the microarray consists ofcDNAs, antisense RNAs are appropriate probes. Therefore, in one aspect,mRNA is used to produce cDNA which, in turn and in the presence offluorescent nucleotides, is used to produce fragments or antisense RNAprobes. These fluorescently labeled probes are incubated with themicroarray so that the probe sequences hybridize to the cDNAoligonucleotides of the microarray. In another aspect, polynucleotidesused as probes can include polynucleotides, fragments, and complementaryor antisense sequences produced using restriction enzymes, PCRtechnologies, and oligolabeling kits (Amersham Pharmacia Biotech) wellknown in the area of hybridization technology.

In another embodiment of the invention, the peptides or polypeptides ofthe invention or functional or immunogenic fragments or oligopeptidesthereof, can be used for screening libraries of compounds in any of avariety of drug screening techniques. The fragment employed in suchscreening may be free in solution, affixed to a solid support, borne ona cell surface, or located intracellularly. The formation of bindingcomplexes, between the peptide or polypeptide and the agent beingtested, may be measured.

One technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe peptide or polypeptide of interest as described in published PCTapplication WO 84/03564. In this method, large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withthe peptide or polypeptide, or fragments thereof, and washed. Boundpeptide or polypeptide is then detected by methods well known in theart. Purified peptide or polypeptide can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another technique, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding the peptide orpolypeptide specifically compete with a test compound for binding to thepeptide or polypeptide. In this manner, the antibodies can be used todetect the presence of a test compound which shares one or more antigenbinding sites with the antibody.

Plant Constructs and Plant Transformants

Of particular interest is the use of the polynucleotides of thisinvention for plant transformation or transfection. Exogenous geneticmaterial may be transferred into a plant cell and the plant cellregenerated into a whole, fertile, or sterile plant. Exogenous geneticmaterial is any genetic material, whether naturally occurring orotherwise, from any source that is capable of being inserted into anyorganism. Such genetic material may be transferred into eithermonocotyledons or dicotyledons including but not limited to the plantsused for animal feed, e.g., feed for sheep, cows, etc.

A variety of methods can be used to generate stable transgenic plants.These include particle gun bombardment (Fromm et al., Bio/Technology8:833-839 (1990)), electroporation of protoplasts (Rhodes et al.,Science 240:204-207 (1989); Shimamoto et al., Nature 338:274-276(1989)), treatment of protoplasts with polyethylene glycol (Datta etal., Bio/Technology, 8:736-740 (1990)), microinjection (Neuhaus et al.,Theoretical and Applied Genetics, 75:30-36 (1987)), immersion of seedsin a DNA solution (Ledoux et al., Nature, 249:17-21 (1974)), andtransformation with T-DNA of Agrobacterium (Valvekens et al., PNAS,85:5536-5540 (1988); Komari, Plant Science, 60:223-229 (1989)). In most,perhaps all plant species, Agrobacterium-mediated transformation is themost efficient and easiest of these methods to use. T-DNA transfergenerally produces the greatest number of transformed plants with thefewest multi-copy insertions, rearrangements, and other undesirableevents.

Many different methods for generating transgenic plants usingAgrobacterium have been described. In general, these methods rely on a“disarmed” Agrobacterium strain that is incapable of inducing tumours,and a binary plasmid transfer system. The disarmed strain has theoncogenic genes of the T-DNA deleted. A Binary plasmid transfer systemconsists of one plasmid with the 23-base pair T-DNA left and rightborder sequences, between which a gene for a selectable marker (e.g. anherbicide resistance gene) and other desired genetic elements arecloned. Another plasmid encodes the Agrobacterium genes necessary foreffecting the transfer of the DNA between the border sequences in thefirst plasmid. Plant tissue is exposed to Agrobacterium carrying the twoplasmids, the DNA between the left and right border repeats istransferred into the plant cells, transformed cells are identified usingthe selectable marker, and whole plants are regenerated from thetransformed tissue. Plant tissue types that have been reported to betransformed using variations of this method include: culturedprotoplasts (Komari, Plant Science, 60:223-229 (1989)), leaf disks(Lloyd et al., Science 234:464-466 (1986)), shoot apices (Gould et al.,Plant Physiology, 95:426-434 (1991)), root segments (Valvekens et al.,PNAS, 85:5536-5540 (1988)), tuber disks (Jin et al., Journal ofBacteriology, 169: 4417-4425 (1987)), and embryos (Gordon-Kamm et al.,Plant Cell, 2:603-618 (1990)).

In the case of Arabidopsis thaliana it is possible to perform in plantgermline transformation (Katavic et al., Molecular and General Genetics,245:363-370 (1994); Clough et al., Plant Journal, 16:735-743 (1998)). Inthe simplest of these methods, flowering Arabidopsis plants are dippedinto a culture of Agrobacterium such as that described in the previousparagraph. Among the seeds produced from these plants, 1% or more haveintegration of T-DNA into the genome.

Monocot plants have generally been more difficult to transform withAgrobacterium than dicot plants. However, “supervirulent” strains ofAgrobacterium with increased expression of the virB and virG genes havebeen reported to transform monocot plants with increased efficiency(Komari et al., Journal of Bacteriology, 166:88-94 (1986); Jin et al.,Journal of Bacteriology, 169:417-425 (1987

Most T-DNA insertion events are due to illegitimate recombination eventsand are targeted to random sites in the genome. However, givensufficient homology between the transferred DNA and genomic sequence, ithas been reported that integration of T-DNA by homologous recombinationmay be obtained at a very low frequency. Even with long stretches of DNAhomology, the frequency of integration by homologous recombinationrelative to integration by illegitimate recombination is roughly 1:1000(Miao et al., Plant Journal, 7:359-365 (1995); Kempin et al.,389:802-803 (1997)).

Exogenous genetic material may be transferred into a plant cell by theuse of a DNA vector or construct designed for such a purpose. Vectorshave been engineered for transformation of large DNA inserts into plantgenomes. Binary bacterial artificial chromosomes have been designed toreplicate in both E. coli and Agrobacterium and have all of the featuresrequired for transferring large inserts of DNA into plant chromosomes.BAC vectors, e.g. a pBACwich, have been developed to achievesite-directed integration of DNA into a genome.

A construct or vector may also include a plant promoter to express thegene or gene fragment of choice. A number of promoters that are activein plant cells have been described in the literature. These include thenopaline synthase (NOS) promoter, the octopine synthase (OCS) promoter,a caulimovirus promoter such as the CaMV 19S promoter and the CaMV 35Spromoter, the figwort mosaic virus 35S promoter, the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter, the sucrose synthasepromoter, the R gene complex promoter, and the chlorophyll a/b bindingprotein gene promoter.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root, or stem, it is preferred that the promotersutilized in the present invention have relatively high expression inthese specific tissues. For this purpose, one may choose from a numberof promoters for genes with tissue- or cell-specific or -enhancedexpression. Examples of such promoters reported in the literatureinclude the chloroplast glutamine synthetase GS2 promoter from pea, thechloroplast fructose-1,6-biphosphatase (FBPase) promoter from wheat, thenuclear photosynthetic ST-LS1 promoter from potato, the phenylalanineammonia-lyase (PAL) promoter and the chalcone synthase (CHS) promoterfrom Arabidopsis thaliana.

Also reported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase (RbcS) promoter from eastern larch(Larix laricina), the promoter for the cab gene, cab6, from pine, thepromoter for the Cab-1 gene from wheat, the promoter for the CAB-1 genefrom spinach, the promoter for the cabiR gene from rice, the pyruvate,orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter forthe tobacco Lhcbl*2 gene, the Arabidopsis thaliana SUC2 sucrose-^(H+)symporter promoter, and the promoter for the thylacoid membrane proteinsfrom spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Otherpromoters for the chlorophyll a/b-binding proteins may also be utilizedin the present invention, such as the promoters for LhcB gene and PsbPgene from white mustard (Sinapis alba). Additional promoters that may beutilized are described, for example, in U.S. Pat. Nos. 5,378,619;5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399;5,633,441; 5,633,435 and 4,633,436, all of which are herein incorporatedin their entirety.

Constructs or vectors may also include, with the coding region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. For example, such sequences havebeen isolated including the Tr7 3′ sequence and the nos 3′ sequence orthe like. It is understood that one or more sequences of the presentinvention that act to terminate transcription may be used.

A vector or construct may also include other regulatory elements orselectable markers. Selectable markers may also be used to select forplants or plant cells that contain the exogenous genetic material.Examples of such include, but are not limited to, a neo gene which codesfor kanamycin resistance and can be selected for using kanamycin, G418,etc.; a bar gene which codes for bialaphos resistance; a mutant EPSPsynthase gene which encodes glyphosate resistance; a nitrilase genewhich confers resistance to bromoxynil, a mutant acetolactate synthasegene (ALS) which confers imidazolinone or sulphonylurea resistance; anda methotrexate resistant DHFR gene.

A vector or construct may also include a screenable marker to monitorexpression. Exemplary screenable markers include a .beta.-glucuronidaseor uidA gene (GUS), an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red colour) in planttissues; a beta-lactamase gene, a gene which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene, a xylE gene which encodes a catecholdioxygenase that can convert chromogenic catechols; an alpha-amylasegene, a tyrosinase gene which encodes an enzyme capable of oxidizingtyrosine to DOPA and dopaquinone which in turn condenses to melanin; analpha-galactosidase, which will turn a chromogenic alpha-galactosesubstrate.

Included within the terms “selectable or screenable marker genes” arealso genes which encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA, small active enzymes detectable in extracellular solution (e.g.,alpha-amylase, beta-lactamase, phosphinothricin transferase), orproteins which are inserted or trapped in the cell wall (such asproteins which include a leader sequence such as that found in theexpression unit of extension or tobacco PR-S). Other possible selectableand/or screenable marker genes will be apparent to those of skill in theart.

Thus, any of the polynucleotides of the present invention may beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promotersenhancers etc. Further any of the polynucleotides encoding a protein orfragment thereof or homologs of the present invention may be introducedinto a plant cell in a manner that allows for expression (e.g.,overexpression) of the protein or fragment thereof encoded by thepolynucleotide.

Computer Related Uses

In one embodiment, a nucleotide or amino acid sequence of the presentinvention can be recorded on computer readable media. This takes intoaccount any medium which can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media. A skilled artisan can readily appreciatehow any of the presently known computer readable mediums can be used tocreate a manufacture comprising computer readable medium having recordedthereon a sequence of the present invention.

A skilled artisan can readily adopt any of the presently know methodsfor recording information on computer readable medium to generatemanufactures comprising the nucleotide sequence information of thepresent invention. A variety of data storage structures are available toa skilled artisan for creating a computer readable medium havingrecorded thereon a nucleotide sequence of the present invention. Thechoice of the data storage structure will generally be based on themeans chosen to access the stored information. In addition, a variety ofdata processor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium.

As non limiting examples, the sequence information can be represented ina word processing text file, formatted in commercially-availablesoftware such as WordPerfect and Microsoft Word, or represented in theform of an ASCII file, stored in a database application, such as DB2,Sybase, Oracle, or the like. A skilled artisan can readily adapt anynumber of data processor structuring formats (e.g. text file ordatabase) in order to obtain computer readable medium having recordedthereon the nucleotide sequence information of the present invention.

By providing the sequence of any SEQ ID NO: herein, or a representativefragment thereof, or any variant thereof, in computer readable form, askilled artisan can routinely access the sequence information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. For example, software can be used to implementthe BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE(Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms, e.g.,on a Sybase system to identify open reading frames (ORFs) which containhomology to ORFs or proteins from other organisms. Such ORFs may beprotein encoding sequences which are useful in producing commerciallyimportant proteins such as enzymes.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantsequences of the M. ruminantium genome. This includes the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based system are suitablefor use in the present invention.

As stated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein a nucleotidesequence of the present invention and the necessary hardware means andsoftware means for supporting and implementing a search means. Thisrefers to memory which can store nucleotide sequence information of thepresent invention, or a memory access means which can accessmanufactures having recorded thereon the nucleotide sequence informationof the present invention. Searching can include one or more programswhich are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means.

Search means are used to identify fragments or regions of the M.ruminantium genome which match a particular target sequence or targetmotif, e.g., antibody targets. A variety of known algorithms aredisclosed publicly and a variety of commercially available software forconducting search means are and can be used in the computer-basedsystems of the present invention. Examples of such software include, butare not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). Askilled artisan can readily recognize that any one of the availablealgorithms or implementing software packages for conducting homologysearches can be adapted for use in the present computer-based systems.

The target sequence can be any DNA or amino acid sequence of six or morenucleotides or two or more amino acids. A skilled artisan can readilyrecognize that the longer a target sequence is, the less likely a targetsequence will be present as a random occurrence in the database. Themost preferred sequence length of a target sequence is from about 10 to100 amino acids or from about 30 to 300 nucleotide residues. However, itis well recognized that searches for commercially important fragments ofthe M. ruminantium genome, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

A target structural motif, or target motif includes any rationallyselected sequence or combination of sequences in which the sequence(s)are chosen based on a three-dimensional configuration which is formedupon the folding of the target motif. There are a variety of targetmotifs known in the art. Protein target motifs include, but are notlimited to, enzyme active sites and signal sequences. Nucleic acidtarget motifs include, but are not limited to, promoter sequences,hairpin structures and inducible expression elements (protein bindingsequences).

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. A preferred format for an output means ranksfragments of the M. ruminantium genome possessing varying degrees ofhomology to the target sequence or target motif. Such presentationprovides a skilled artisan with a ranking of sequences which containvarious amounts of the target sequence or target motif and identifiesthe degree of homology contained in the identified fragment.

A variety of comparing means can be used to compare a target sequence ortarget motif with the data storage means to identify sequence fragmentsof the M. ruminantium genome. In particular aspects, software can beused implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol.Biol. 215:403-410. (1990)) and to identify open reading frames. Askilled artisan can readily recognize that any one of the publiclyavailable homology search programs can be used as the search means forthe computer-based systems of the present invention

The computer system may include a processor connected to a bus. Alsoconnected to the bus may be a main memory (preferably implemented asrandom access memory, RAM) and a variety of secondary storage devices,such as a hard drive and a removable medium storage device. Theremovable medium storage device may represent, for example, a floppydisk drive, a CD-ROM drive, a magnetic tape drive, etc. A removablestorage medium (such as a floppy disk, a compact disk, a magnetic tape,etc.) containing control logic and/or data recorded therein may beinserted into the removable medium storage device. The computer systemmay include appropriate software for reading the control logic and/orthe data from the removable medium storage device once inserted in theremovable medium storage device.

A sequence of the present invention may be stored in a well known mannerin the main memory, any of the secondary storage devices, and/or aremovable storage medium. Software for accessing and processing thegenomic sequence (such as search tools, comparing tools, etc.) reside inmain memory during execution.

EXAMPLES

The examples described herein are for purposes of illustratingembodiments of the invention. Other embodiments, methods, and types ofanalyses are within the scope of persons of ordinary skill in themolecular diagnostic arts and need not be described in detail hereon.Other embodiments within the scope of the art are considered to be partof this invention.

Example 1: Materials and Methods Strain Information and GrowthConditions

Methanobrevibacter ruminantium M1^(T) (DSM1093) was obtained from theGerman Collection of Microorganisms and Cell Cultures (DSMZ),Braunschweig, Germany. The original description of Methanobacteriumruminantium was made by Smith and Hungate (Smith & Hungate, 1958) andthe genus designation later changed to Methanobrevibacter (Balch et al.,1979). Methanobrevibacter ruminantium M1^(T) (DSM1093) was isolated frombovine rumen contents by Bryant (Bryant, 1965). It is designated theneotype strain for this species because the original strain of Smith andHungate was not maintained. M. ruminantium strain M1^(T) was routinelygrown in basal medium (Joblin et al., 1990) with added trace elements(Balch et al., 1979), (BY⁺ medium), with H₂ plus CO₂ (4:1) at 180 kPaoverpressure. The culture tubes were incubated on their sides, at 39° C.in the dark, on a platform shaken at 200 rpm.

Co-Culture of M. ruminantium and Butyrivibrio proteoclasticus

M1 was grown in co-culture with Butyrivibrio proteoclasticus B316^(T)(DSM14932) to examine gene expression under rumen-like conditions.Eighteen pure cultures of M1 were grown in BY⁺ medium with H₂ plus CO₂(4:1) at 180 kPa overpressure in 100 ml volumes in 125 ml serum bottlessealed with blue butyl septum stoppers and aluminium seals (BellcoGlass, Vineland, N.J., USA). When the cultures reached mid-exponentialphase, as measured by optical density at 600 nm (Ultrospec 1100 proUV/Vis spectrophotometer, Amersham Biosciences, Little Chalfont,Buckinghamshire, UK) they were flushed with O₂-free 100% CO₂ gas untilH₂ was not detectible by gas chromatography. All 18 cultures weresupplemented with oat spelt xylan (Sigma-Aldrich, St. Louis, Mo., USA)to 0.2% (w/v) final concentration, then nine of the cultures wereinoculated with 0.5 ml of a late-exponential phase culture of B.proteoclasticus. The other nine were re-pressurized to H₂ plus CO₂ (4:1)at 180 kPa overpressure. Three further serum bottles of BY⁺ mediumsupplemented with 0.2% (w/v) xylan were also inoculated with anequivalent inoculum of B. proteoclasticus. Growth in the co-culture wasmonitored periodically by Thoma slide enumeration (Webber ScientificInternational Ltd., Teddington, England). Mid-exponential phaseco-cultures and monocultures were harvested by centrifugation (10,000×g,5 min at 4° C.), and the cell pellets resuspended in 10 ml of BY⁺ medium(+0.2% [w/v] xylan) and 20 ml of RNAprotect (Qiagen, Hilden, Germany).These were incubated for 5 min at room temperature, and were immediatelyprocessed for RNA extraction

Microarray Analyses

RNA Isolation, cDNA Synthesis and Labelling.

Cells of M1 and B. proteoclasticus from mono- or co-cultures prepared asdescribed above, were pelleted by centrifugation (5,000×g, 10 min roomtemperature), air-dried and frozen under liquid N₂. Frozen pellets wereground in a sterile pre-chilled (−20° C.) mortar and pestle under liquidN₂, and the ground samples resuspended in excess TRIzol (Invitrogen,Carlsbad, Calif., USA). The mixtures were incubated at 20° C. for 5 min.Chloroform (200 μl) was then added, mixed vigorously, and incubated fora further 3 min. The samples were centrifuged (12,000×g, 15 min, 4° C.)and the aqueous phases transferred to fresh tubes, mixed with 0.5volumes isopropanol and incubated at 20° C. for 10 min to precipitatethe RNAs. Precipitated RNAs were pelleted by centrifugation (12,000×g,10 min, 4° C.), the supernatants removed and the RNAs washed with 5 mlof 75% (v/v) ethanol before being re-pelleted by centrifugation. Ethanolwas removed, the pellets air dried on ice and finally each resuspendedin 1 ml of diethyl pyrocarbonate (DEPC) treated Milli-Q water. The RNAswere further purified using an RNeasy Midi kit (Qiagen, Hilden, Germany)and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies,Santa Clara, Calif., USA) following the respective manufacturer'sinstructions. cDNA synthesis, labeling and purification were carried outusing the Invitrogen cDNA labelling purification kit, while the Cy3 andCy5 dyes were from GE Healthcare (Uppsala, Sweden).

Quantification of Co-Culture mRNA.

The relative quantities of RNAs contributed by each organism to theco-culture samples were determined by quantitative PCR of the B.proteoclasticus butyryl-CoA dehydrogenase (bcd) gene (using primersbcdqfp: tgagaagggaacacctggat; SEQ ID NO: 7586, and bcdqrp:ttgctcttccgaactgctt; SEQ ID NO: 7587), and the M1 gene encodingN⁵,N¹⁰-methenyl-H₄MPT cyclohydrolase (mch) (using primers mchqfp:gtattgcctggtgaagatgt; SEQ ID NO: 7588 and mchqrp: gtcgatttggtagaagtca;SEQ ID NO: 7589). Homologs of both genes have previously been shown tobe constitutively expressed in closely related species (Reeve et al.,1997; Asanuma et al., 2005). The mono-culture RNAs were then combined inequal proportions to normalise mRNA abundance with their co-culturereplicates.

Probe Synthesis and Slide Printing.

Oligonucleotide 70 mer probes were designed against the draft genomes ofM1 and Butyrivibrio proteoclasticus B316^(T) using ROSO software(Reymond et al., 2004) and synthesised by Illumina (San Diego, Calif.,USA). Oligonucleotides were spotted onto epoxy-coated slides (Corning,Lowell, Mass., USA) using an ESI robot (Engineer Service Inc., Toronto,Ontario, Canada).

Microarray Hybridization and Scanning.

Microarrays were replicated 6 times (3 biological replicates pertreatment, each with a dye swap) and each gene was represented on thearray 3 to 7 times. Microarray slides were pre-warmed in microarrayprehybridization buffer (50° C. for 30 min), and transferred intohybridization chambers (Corning, Lowell, Mass., USA) and lifter coverslips (Erie Scientific, Portsmouth, N.H., USA) were laid over the probeareas. Samples of RNA to be compared (e.g., Cy3 co-culture versuscombined Cy5 individual mono-cultures) were combined, denatured at 95°C. for 10 min, and mixed with 60 μl of pre-warmed (68° C.) Slide Hybbuffer #1 (Ambion, Austin, Tex., USA). The mixture was loaded onto theslide, the hybridization chamber sealed, and incubated in a water bathat 50° C. for 24 h. Following hybridization, the slides were washed byvigorous shaking by hand in pre-warmed (50° C.) wash solutions 1 to 3(wash solution 1: 10% SDS, 2×SSC; wash solution 2: 1×SSC; wash solution3: 0.1×SSC), 7 min per wash in aluminium foil-covered Falcon tubes(Becton, Dickinson and Co. Sparks, Md., USA). Following the third wash,the slides were dried by low speed centrifugation (1,500×g, 4 min)followed by incubation for 20 min in a 37° C. vacuum oven (Contherm,Wellington, NZ) in the dark. Microarray slides were scanned using aGenePix@ Professional 4200 scanner and GenePix Pro 6.0 software(Molecular Devices, Sunnyvale, Calif., USA) and analysed using the Limmapackage in Bioconductor (Smyth, 2005). Genes with an up- ordown-regulation of 2 fold or greater and an FDR value <0.05 were deemedstatistically significant.

Growth Experiments to Test Effects of PeiR and Alcohols

M1 was grown in medium RM02 in anaerobic culture tubes (16 mm internaldiameter, 18 mm outer diameter, 150 mm long; Bellco Glass, Vineland,N.J., USA), essentially as described by Balch and Wolfe, 1976. Themineral salts base of RM02 contained (per litre of medium): 1.4 g ofKH₂PO₄, 0.6 g of (NH₄)₂SO₄, 1.5 g of KCl, 1 ml trace element solutionSL10 (Widdel et al., 1983), 1 ml of selenite/tungstate solution (Tschechand Pfennig, 1984) and 4 drops of 0.1% (w/v) resazurin solution. Thissolution was mixed and then boiled under O₂-free 100% CO₂, before beingcooled in an ice bath while it was bubbled with 100% CO₂. Once themedium was cool, 4.2 g of NaHCO₃ and 0.5 g of L-cysteine.HCl.H₂O wasadded per litre. The medium was dispensed into the culture tubes whilebeing gassed with 100% CO₂, at 9.5 ml of medium per tube, and the tubessealed with blue butyl septum stoppers and aluminium seals (Bellco),with a headspace of 100% CO₂. These tubes were sterilised by autoclavingfor 20 min at 121° C. Before use, the tubes were stored in the dark forat least 24 h. Sodium acetate (20 mM final conc.), sodium formate (60 mMfinal conc.), coenzyme M (10 μM final conc.), and vitamin-supplementedclarified rumen fluid were added to sterile media, before inoculationwith 0.5 ml of an actively growing culture of M. ruminantium, thengassed with H₂ plus CO₂ (4:1) to 180 kPa overpressure. In someexperiments, the formate was omitted, and alcohols were added, as notedin the experimental descriptions accompanying the results. The culturetubes were incubated on their sides, at 39° C. in the dark, on aplatform shaken at 200 rpm.

To prepare the clarified rumen fluid, rumen contents were collected froma ruminally-fistulated cow that had been fed hay for 48 h after being ona rye-grass clover pasture. Feed was withheld from the animal overnightand rumen contents collected the next morning. The material was filteredthrough a single layer of cheesecloth and then fine particulate materialremoved by centrifugation at 10,000×g for 20 min. The supernatant wasstored at −20° C. Before further use, it was thawed, and anyprecipitates removed by centrifugation at 12,000×g for 15 min. Thesupernatant was bubbled for 10 min with 100% N₂ gas, before beingautoclaved under 100% nitrogen for 15 min to remove viruses. Thefollowing was then added per 100 ml of rumen fluid while stirring underair: 1.63 g of MgCl₂.6H₂O and 1.18 g of CaCl₂.2H₂O. The resulting heavyprecipitate was removed by centrifuging at 30,000×g and 4° C. for 60min. The supernatant was designated the clarified rumen fluid. Two gramsof yeast extract powder was added, and the mixture then bubbled with N₂gas for 15 min, before being transferred to a N₂-flushed sterile serumvial through a 0.2-μm pore size sterile filter using a syringe andneedle.

Two ml of Vitamin 10 concentrate was then added per 100 ml ofpreparation by syringe and needle. Vitamin 10 concentrate contained 1000ml of distilled water, 40 mg of 4-aminobenzoate, 10 mg of D-(+)-biotin,100 mg of nicotinic acid, 50 mg of hemicalcium D-(+)-pantothenate, 150mg of pyridoxamine hydrochloride, 100 mg of thiamine chloridehydrochloride, 50 mg of cyanocobalamin, 30 mg of D,L-6,8-thioctic acid,30 mg of riboflavin and 10 mg of folic acid. After preparation, thesolution was well mixed and then bubbled with N₂ gas for 15 min, beforebeing transferred to a N₂-flushed sterile serum vial through a 0.2 μmpore size sterile filter using a syringe and needle.

Growth of M1 was followed by measuring the culture density at 600 nm byinserting the tubes directly into an Ultrospec 1100 pro UV/Visspectrophotometer (Amersham Biosciences, Little Chalfont,Buckinghamshire, UK). Tubes containing 10 ml of medium RM02 wereinoculated with 0.5 ml of an actively growing culture of M1, then gassedwith H₂ plus CO₂ (4:1) to 180 kPa overpressure. Additions of PeiR in 0.1ml of buffer (20 mM 3-[N-morpholino]propane sulfonic acid: 1 mMdithiothreitol:0.3 M NaCl, 20% glycerol [v/v], pH 7.0 with NaOH), 0.1 mlof buffer only, or 0.1 ml of chloroform were made when the cultures hadgrown to mid-exponential phase (optical density at 600 nm [OD₆₀₀]˜0.1,16 mm path length). In the experiments testing the effects of PeiRaddition, the culture densities were mathematically normalised to anOD₆₀₀ of 0.1 at the time the additions were made, and all other readingscorrected by the same ratio. This was done to remove the effect of lackof absolute synchronicity between cultures, a common phenomenon whenculturing methanogens. This normalisation was not done for experimentstesting the utilisation of alcohols. Methane was measured by gaschromatography, taking a 0.3 ml sample from the culture headspace, atthe pressure in the culture tube, and injecting it into an Aerograph 660(Varian Associates, Palo Alto, Calif., USA) fitted with a Porapak Q80/100 mesh column (Waters Corporation, Milford, Mass., USA) and athermal conductivity detector operated at 100° C. The column wasoperated at room temperature with N₂ as the carrier gas at 12 cm³/min.

DNA Extraction

Genomic DNA was extracted from M1 grown on BY⁺ medium with H₂ plus CO₂(4:1), using the liquid N₂ freezing and grinding method (Jarrell et al.,1992). Briefly, M1 cultures were harvested by centrifugation at 27,000×gfor 20 min at 4° C. and cell pellets combined and placed into 40 mlOakridge centrifuge tubes (Thermo Fisher Scientific, Inc.). The cellswere frozen at −20° C. and kept frozen for at least 4 days. The frozencell pellets were placed in a sterile, pre-cooled (−85° C.) mortar andliquid N₂ poured over the pellet. After the N₂ had evaporated, thepellet was ground to a powder with a sterile glass rod. Immediately, 0.5ml of TES buffer (10 mM Tris-HCl:1 mM EDTA:0.25 M sucrose, pH 7.5) wasadded to the powdered cell pellet and mixed gently into a slurry. Sodiumdodecyl sulfate was added to a final concentration of 1% (w/v) andProteinase K (Roche Diagnostics, Mannheim, Germany) added to a finalconcentration of 50 μg/ml. The mixture was incubated at 60° C. for 30min. NaCl was added to a final concentration of 0.5 M and the lysate wasplaced on ice for 1 h. The lysate was centrifuged at 25,000×g for 15 minat 4° C. and the supernatant recovered carefully. An equal volume ofcold (4° C.) isopropanol was added to the supernatant, and theprecipitated DNA was collected by centrifugation at 12,000×g for 10 minat room temperature and re-dissolved in TE buffer (10 mM Tris-HCl:1 mMEDTA, pH 7.5). The DNA was treated with RNase (10 μg/ml),(Sigma-Aldrich) for 30 min at 37° C., and extracted twice with an equalvolume of phenol/chloroform/isoamyl alcohol (25:24:1) and twice with anequal volume of chloroform alone. NaCl was added to a finalconcentration of 0.5 M and the DNA precipitated by adding 2.5 volumes ofcold (4° C.) ethanol. The precipitated DNA was collected bycentrifugation at 14,000×g for 10 min at 4° C. and re-dissolved in TEbuffer.

Pulsed-Field Gel Electrophoresis (PFGE)

Standard PFGE protocol involves embedding cells in agarose and lysiswith lysozyme and/or proteases, but this was not possible with M1because its pseudomurein-containing cell wall was resistant to lysis bycommercially available enzymes. In order to overcome this, the cellpellet from a centrifuged 50 ml culture was frozen with liquid N₂ andvery gently ground in a pestle and mortar to damage the cell wall. Theground material was allowed to thaw, 2 ml of 1 M NaCl plus 10 mM Tris(pH 7.6) was added and 300 I aliquots were mixed with an equal volume of2% (w/v) low melt agarose (Bio-Rad Laboratories, Hercules, Calif., USA).Embedded cells were treated with 0.1 mg ml⁻¹ Proteinase K in lysisbuffer (50 mM Tris-HCl:50 mM EDTA:1% [w/v] sarkosyl, pH 8.0) at 50° C.for up to 24 h. The agarose plugs were washed twice with sterile waterand three times with TE buffer (10 mM Tris-HCl:1 mM EDTA, pH 8.0) beforestorage in 10 mM Tris-HCl:100 mM EDTA (pH 8.0) at 4° C. DNA embedded inagarose was digested for 16 h with 1.0 U of ApaI, BssHII or Mlul (NewEngland Biolabs, Beverly, Mass., USA) in 100 μl of restriction enzymebuffer, loaded into wells of 1% (w/v) agarose gels (SeaKem Gold agarose,Cambrex Bio Science, Rockland, Me., USA), and run at 200 V for 20 h at14° C. in 0.5× Tris-borate buffer using a CHEF DR III PFGE apparatus andmodel 1000 mini chiller (Bio-Rad). Double-digest combinations of theseenzymes were digested and run in the same way. DNA was visualized bystaining with ethidium bromide and the image captured using a Gel Doc1000 system (Kodak Gel Logic 200 Imaging System, Eastman Kodak,Rochester, N.Y., USA).

Genome Sequencing, Assembly and Validation

The genome sequence of M1 was determined using a whole genome shotgunstrategy (Agencourt Biosciences, USA) and a pyrosequencing approach(Macrogen, USA). A hybrid assembly (Goldberg et al., 2006) was performedutilising the Staden package (Staden & Bonfield, 2000), Phred (Ewing etal., 1998), Phrap (hypertext transfer protocol://www.phrap.org), Paracel(Paracel Inc.) and Repeatmasker (hypertext transferprotocol://repeatmasker.org) resulting in a 27 contig assembly. Gapswere closed using additional sequencing by PCR-based techniques. Qualityimprovement of the genome sequence was performed using standard PCR toensure correct assembly and the resolution of any remainingbase-conflicts. Assembly validation was confirmed by pulsed-field gelelectrophoresis (see above).

Genome Analysis and Annotation

A GAMOLA (Altermann and Klaenhammer, 2003) Artemis (Rutherford et al.,2000) software suite was used to manage genome annotation.Protein-encoding open reading frames (ORFs) were identified using theORF-prediction program Glimmer (Delcher et al., 1999) and BLASTX (Gish &States, 1993). A manual inspection was performed to verify or, ifnecessary, redefine the start and stop of each ORF. Assignment ofprotein function to ORFs was performed manually using results from thefollowing sources; BLASTP (Altschul et al., 1990) to both anon-redundant protein database provided by the National Centre forBiotechnology Information (NCBI) (Sayers et al., 2009) and clusters oforthologous groups (COG) (Tatusov et al., 2000) database. HMMER (Eddy,1998) was used to identify protein motifs to both the PFAM (Finn et al.,2008) and TIGRFAM (Haft et al., 2003) libraries. TMHMM (Krogh et al.,2001; hypertext transfer protocol://www.cbs.dtu.dk/services/TMHMM/) wasused to predict transmembrane sequences, and SignalP (Bendtsen et al.,2004) was used for the prediction of signal peptides.

Ribosomal RNA genes were detected on the basis of BLASTN searches to acustom GAMOLA ribosomal database. Transfer RNA genes were identifiedusing tRNAscan-SE (Lowe & Eddy, 1997). Miscellaneous-coding RNAs wereidentified using the Rfam database utilizing the INFERNAL softwarepackage (Eddy, 2002). Insertion sequence elements were identified usingRepeatfinder (Volfovsky et al., 2001) and BLAST and annotated manually.Genome atlas visualisations were constructed using GENEWIZ (Jensen etal., 1999). Horizontal gene transfer studies were performed usingDarkhorse (Podell & Gaasterland, 2007), GC % content (Rice et al., 2000)and the Codon Adaptation Index (Sharp & Li, 1987). A BLAST analysis wasperformed against the arCOG (Makarova et al., 2007) database. Analysisof non-ribosomal peptide synthetases (NRPSs) was performed usingNRPSpredictor (Rausch et al., 2005). An LPxTG-HMM (Boekhorst et al.,2005) was used for the identification of LPxTG motifs. Metabolic pathwayreconstructions were performed using Pathway Voyager (Altermann &Klaenhammer, 2005) and the KEGG (Kyoto Encyclopedia of Genes andGenomes) database (Kanehisa & Goto, 2000) combined with an extensivereview of the literature. Identification of open reading frames (ORFs)as vaccine and drug targets was performed as described. Genome sequencesused in comparative studies were downloaded from the National Centre forBiotechnology Information (NCBI) FTP website and are listed in Table 10,below.

Genome sequence was prepared for NCBI submission using Sequin (Benson etal., 2009). The adenine residue of the start codon of the Cdc6-1(mru0001) gene was chosen as the first base for the M1 genome. For GCskew and synteny analysis, the sequences of genomes of other members ofthe order Methanobacteriales were rotated to begin at the same location.GC skew analysis was performed by circular diagram.pl (Rutherford, K,Sanger Centre software) and synteny plots were generated using MUMmer3.0(Delcher et al., 2003).

Vaccine Target ORF Identification

Vaccine targets are likely to be surface exposed or membrane associatedand conserved among methanogens or archaeal species. Methanogens are theonly known resident archaea in the rumen and therefore archaeal specificcandidates were not omitted from target lists, likewise when present,sequence homology to proteins associated with known vaccine or drugdesign remained a strong element for target selection regardless ofother criteria. To identify the surface-exposed or membrane-associatedORFs of M1 a combination of methods was utilized. To date, there is nosignal peptide model for archaea. There are simply too fewexperimentally verified secretory proteins available for Archaea totrain a specific model. For this reason ORF sequences were analysed forthe presence of signal peptides using SignalP Version 3.0 (Bendtsen etal., 2004) trained against the Gram-positive, Gram-negative andEukaryotic models and the results combined. SignalP-HMM (hidden markovmodels) was used to discriminate between signal peptide and non-signalpeptide ORFs whereas SignalP-NN (neural networks) was utilized for theprediction of cleavage sites as described by Emanuelsson et al., 2007(Emanuelsson et al., 2007). TMHMM (Krogh et al., 2001; hypertexttransfer protocol://www.cbs.dtu.dk/services/TMHMM) was used for theprediction of transmembrane domains and (Nakai & Horton, 1999) PSORTtrained on a Gram-positive model was used to predict a protein'ssubcellular localization. BLASTP results were analyzed to identifymethanogen specific ORFs.

Chemogenomics Target ORF Identification

Three different approaches were utilized to identify candidatechemogenomics targets.

Metabolic Profiling Analyses.

Several factors were taken into consideration when performing thisanalysis. Utilizing the metabolic reconstruction of M1 and an extensivereview of the literature, archaeal- or methanogen-specific enzymes, orenzymes with sufficient structural or biochemical differences comparedto their bacterial or eukaryl counterparts were identified. Somemethanogen enzymes or pathways that have been previously targeted byresearchers for inhibition demonstrating the essentiality of certainenzymes/pathways were also taken into consideration. In addition, a fewenzymes which represent key enzymes to several pathways or are wellknown validated targets in pathogenic bacteria or parasites, whilststill retaining sufficient sequence divergence to potentially be able tobe targeted effectively were also included. Most of the cell wallenzymes are listed as the majority of successful antibiotics that havebeen developed against pathogenic bacteria target cell wallbiosynthesis. Methanobacterial cell wall synthesis, despite apparentlysharing some common enzymes (e.g., mur ligases) is widely divergent inbiochemical terms from bacterial cell wall synthesis and the homologousenzymes share only limited sequence homology. The degree to which strainM1, or other rumen methanogens, are able to utilize amino acids,vitamins, or purine or pyrimidine compounds in rumen fluid is underinvestigation.

Functional Genome Distribution (FGD).

A FGD analysis (Altermann 2009, manuscript in preparation) was performedusing 26 publicly available draft and complete methanogen genomesequences (dbMethano, Table 10). In contrast to an evolutionaryphylogeny, FGD analyzes the functional relationship between microbesbased on their predicted ORFeomes. FGD is a comparative genomicsapproach to genome-genome comparisons, emphasizing functionalrelationships rather than ancestral lineages. Briefly, pooled ORFeomesare subjected to all-vs-all analyses, evaluating the level and qualityof amino-acid similarities between individual ORFs pairings. Individualresults for each genome-genome combination are then combined into asymmetrical distance matrix and can be visualized using the UnweightedPair Group Method with Arithmetic mean (UPGMA) method (Sneath and Sokal,1973) Numerical Taxonomy. Freeman, San Francisco. Strain and clusterconserved and specific gene sets were mined based on respective BLASTe-values, using custom developed software.

Differential Blast Analysis (DBA).

The reference genome of M1 was subjected to analysis against two BLASTPdatabases using GAMOLA (Altermann and Klaenhammer, 2003). The firstamino-acid database employed all methanogen ORFeomes used in the FGDanalysis (dbMethano), while the second database was comprised of thenon-redundant database (nr) as provided by NCBI, excluding hits togenera used in dbMethano. E-values of best BLASTP hits for both databasesets were consolidated into an empirically determined e-value trustlevel range ([T_(e-value)]) and their respective differential calculatedas follows: Δ=(T_(nr)−T_(dbMethano)). Results were visualized on agenome atlas using Genewiz (Jensen et al., 1999) and software developedin-house.

Peptide Vaccine Methods

The use of synthetic peptides to raise antibodies against predicted M1surface proteins was investigated. The M1 proteins encoding themembrane-spanning subunits of tetrahydromethanopterinS-methyltransferase (MtrCDE, mru1921, 1922 and 1923), adhesin-likeproteins (mru2049, 0842, 0143 and 2048) and a magnesium chelatasesubunit H (BchH, mru2047) containing N-terminal and C-terminal TMHs,were analysed to identify extracellular peptide sequences which mightserve as potential antibody binding sites. Nine suitable peptidesequences from extracellular regions of these eight proteins wereidentified and used to guide the manufacture of the correspondingsynthetic peptides. Each peptide was coupled to the Keyhole Limpethemocyanin (KLH) protein via an additional N- or C-terminal cysteineresidue and a maleimidocaproyl-N-hydroxysuccinimide linker and used toraise antibodies in sheep (Invitrogen, USA). The conjugated peptides(200 μg) were injected intradermally (ID) into sheep (1-3 yr age) inComplete Freund's Adjuvant (CFA) at 10-15 sites on day 0, and secondaryboosters in CFA were given on day 14. Six ID injections of 200 μgKLH-peptide in Incomplete Freund's Adjuvant at 10-15 sites were given atdays 28, 56, 70, 84, 98 and 112. Test bleeds (2-5 ml) were taken on days42, 56, 84, and 112 for ELISA analyses.

Antibody titer was determined with an ELISA with Peptide-GGG (goat gammaglobulin) bound in solid phase (0.1 μg/100 μl/well) on high binding 96well plates. The serum was first diluted 50-fold and then furtherdiluted in 2-fold serial dilutions. The ELISA titer is the estimateddilution factor that resulted in an OD₄₀₅ nm of 0.2 and is derived fromnonlinear regression analysis of the serial dilution curve. Detectionwas obtained using an HRP (horseradish peroxidase)-conjugated secondaryantibody and ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonicacid). In the antibody-binding experiment M1 cells (40 μl of cells in 2ml of sodium carbonate buffer) were immobilised on Maxisorp ELISA platesand antibody binding was determined by ELISA. Serum samples were diluted1/20 in diluent (1% w/v casein in PBS Tween 20 (g/l NaCl, 8.0; KCl, 0.2;Na2HPO4, 1.15; KH2PO4, 0.2; pH 7.2-7.4; Tween 20, 0.5 ml) and incubatedat room temperature for 1 hr. Plates were washed 6 times with PBS Tween20 and conjugate (donkey anti-sheep/goat IgG HRP, 50 μl/well of a 1/5000diluted solution) and substrate (3, 3′,5,5′ tetramethyl benzidine, 50μl/well) were added. After incubation at room temperature in the darkfor 15 min, stop solution (50 μl/well of 0.05 M H2SO4) was added andOD₄₅₀ nm readings were taken.

Accession Numbers and Sequence Listing

The nucleotide sequence of the M. ruminantium M1 chromosome has beendeposited in GenBank under Accession Number CP001719. Microarray datahas been submitted to the Gene Expression Omnibus (GEO) in accordancewith MIAME standards under GEO Accession Number GSE18716. The specificM. ruminantium sequences are disclosed herewith as a text file (.txt)for the sequence listing. The contents of sequence listing are herebyincorporated by reference herein in their entirety.

Example 2: Results General Genome Characteristics

The genome sequence of M1 consists of a single 2.93 megabase (Mb)circular chromosome, the assembly of which has been verified bypulsed-field gel electrophoresis (FIG. 6). The general features of theM1 genome compared to other genomes of species within the orderMethanobacteriales are summarized in Table 1, below, and FIG. 10. M1 hasthe largest genome of the Methanobacteriales sequenced to date. Thisincreased genome size is due in part to a lower overall coding density,but also to a large number of genes encoding surface adhesin-likeproteins, the presence of a prophage, and a variety of genes unique tothe M1 genome. M1 encodes 2217 open reading frames (ORFs) and afunctional classification of each ORF is presented in Table 9, below,and FIG. 15. Genomes of the Methanobacteriales display a GC skew similarto bacterial chromosomes (Lobry, 1996) (FIG. 2) and an X-shaped syntenypattern that is characteristic of moderately diverged genomes (FIG. 3).Analysis of potential horizontal gene transfer (HGT) events in M1identified a number of genes which show high sequence similarity tonon-methanogens, typically from members of the bacterial phylumFirmicutes (Table 4, below, and FIG. 16). These potential HGT events canbe visualized in a BLAST heat map analysis (FIG. 16). Several approacheswere used to define potential gene targets for CH₄ mitigation from M1.An integral part of this process was the analysis of genes which areconserved across methanogen genomes (FIG. 13 and Table 8, below).Coupled with this, chemogenomic targets (FIG. 1a ) were selected on thebasis of detailed metabolic analysis (Table 2, below), while potentialvaccine targets (FIG. 1b ) were chosen from proteins predicted to beassociated with the M1 cell surface. Examination of the M1 genome hasrevealed a number of features and targets that could lead to aneffective enteric methane mitigation technology, and these are discussedbelow.

Growth and Methanogenesis

Many of the enzymes involved in the methanogenesis pathway are stronglyconserved and found only among methanogens, and therefore present goodtargets for CH₄ mitigation technologies. Although this pathway has beenwell studied in methanogens from a range of other environments (Thaueret al., 2008), the M1 genome shows for the first time details of thispathway in a rumen methanogen. M1 can grow with H₂+CO₂ and formate(Smith & Hungate, 1958) and encodes the enzymes, and most of thecofactors, required for conversion of these substrates through tomethane according to the metabolic scheme presented in FIG. 11.Consistent with this hydrogenotrophic lifestyle, M1 lacks themethanophenazine-reducing [Ni-Fe] hydrogenase (VhoACG) andmethanophenazine-dependant heterodisulphide reductase (HdrDE) found inmethanophenazine-containing species within the order Methanosarcinales(Abken et al., 1998).

Surprisingly, M1 has two NADPH-dependent F₄₂₀ dehydrogenase (npdG1, 2)genes and three NADP-dependent alcohol dehydrogenase (adh1, 2 and 3)genes. In some methanogens, these enzymes allow growth on ethanol orisopropanol via NADP⁺-dependent oxidation of the alcohol coupled to F₄₂₀reduction of methenyl-H₄MPT to methyl-H₄MPT (Berk & Thauer, 1997) (FIG.11). M1 is reported as not being able to grow on ethanol or methanol(Smith & Hungate, 1958), although a ciliate-associated M.ruminantium-like isolate was able to use isopropanol to a limited degreebut data were not presented (Tokura et al., 1999). Our attempts to growM1 on alcohols indicate that ethanol and methanol stimulate growth inthe presence of limiting amounts of H₂+CO₂, but they do not supportgrowth when H₂ is absent (FIG. 14). M1 does not contain homologues ofthe mta genes known to be required for methanol utilization in othermethanogens (Fricke et al., 2006). The adh genes may play a role inalcohol metabolism but the mechanism is under assessment.

Hydrogenotrophic methanogens usually encode a methyl coenzyme reductaseII (mcrII or mrt), an isoenzyme of the methyl CoM reductase I (mcrI)enzyme which is differentially regulated during growth (Reeve et al.,1997) to mediate methane formation at high partial pressures of H₂.Interestingly, M1 does not encode a mcrII system. In the rumen,methanogens depend on fermentative microbes to supply H₂, usually atvery low concentrations, and M1 appears to have adapted its lifestylefor growth at low levels of H₂ using the mcrI system only.

To examine the expression of genes involved in methanogenesis, in thepresence of a H₂-forming rumen bacterium, M1 was grown in co-culturewith Butyrivibrio proteoclasticus B316 (Moon et al., 2008) in a mediumcontaining xylan as the sole carbon source, and gene expression wasanalysed by microarrays.

Formylmethanofuran dehydrogenase (fwdA), methyl CoM reductase (mcrBCDG),methyl viologen-reducing hydrogenase (mvhG), and H₄MPT methyltransferase(mtrABCH) were significantly up-regulated (>2 fold) in the co-culturecompared to the monoculture of M1 grown with H₂+CO₂ (Table 5, below).Interestingly, formate utilisation (fdhAB) genes were also up-regulated,suggesting that formate formed by B. proteoclasticus was an importantmethanogenic substrate transferred during this syntrophic interaction.

Genes encoding enzymes in the methanogenesis pathway that are potentialtargets are highlighted in FIG. 11. Several methanogenesis markerproteins found in methanogen genomes, with hypothetical function, werealso included in the target list. Many of the enzyme subunit targets arepredicted to be within the cell cytoplasm, and therefore best pursuedvia a chemogenomics approach (FIG. 11). However, several, subunitsincluding those of the Aha, Eha, Ehb and Mtr enzyme complexes, aremembrane-located and may be suitable as vaccine targets. Mtr catalysesthe transfer of the methyl group from methyl-H₄MPT to CoM and couplesthis to the efflux of Na⁺ ions (Reeve et al., 1997). Three of the Mtrsubunits (MtrEDC) are predicted to be membrane-spanning in M1 and ineach of the membrane-spanning regions the transmembrane helices havepeptide loops located outside the cell membrane. These loops arepotential antibody binding sites. Synthetic peptides corresponding tothe loop regions of MtrE, MtrD and MtrC have been coupled to a carrierprotein and used as antigens to vaccinate sheep. The resulting immunesera were shown to bind specifically to immobilized M1 cells (FIG. 17),demonstrating the feasibility of such a peptide-directed reversevaccinology approach.

Analysis of the M1 genome has helped explain the growth requirements ofM1 for acetate, 2-methylbutyrate and co-enzyme M (CoM) (Bryant et al.,1971). Acetate is required for cell carbon biosynthesis after activationto acetyl CoA (acs, acsA), followed by reductive carboxylation topyruvate (porABCDEF, Table 9, below). Reductive carboxylation of2-methylbutyrate is probably the route for isoleucine biosynthesis(Robinson & Allison, 1969), as M1 lacks a gene encoding a homoserinekinase needed for the usual pathway from threonine (Table 9, below).Exogenously supplied CoM is essential for M1 growth as two genes neededin the CoM biosynthetic pathway, phosphosulfolactate synthase (comA) andsulfopyruvate decarboxylase (comDE) (Graham et al., 2002), are missingin M1 (FIG. 11).

Cell Envelope

The methanogen cell envelope serves as the interface between theorganism and its rumen environment, and as such represents a key areafor the identification of vaccine and drug targets. The main structuralcomponent of the cell envelope of M1 (FIG. 12), as with otherGram-positive methanogens, is pseudomurein. This is structurallyanalogous, but chemically different, to peptidoglycan, which performsthe comparable function in bacteria (König et al., 1994). Bacterialpeptidoglycan biosynthesis has long been a major target ofantimicrobials but these compounds are largely ineffective againstpseudomurein-containing cells (Kandler & König, 1998). The pathway forpseudomurein biosynthesis and its primary structure have been proposed(Kandler & König, 1998), but the enzymes involved have not beencharacterized. Our genomic analysis has identified several genesencoding enzymes likely to be involved both in the intracellularbiosynthesis of the pseudomurein precursors and the processes involvedin exporting and assembling these into the cell wall (FIG. 5).

The original description of M. ruminantium reported the existence of acapsule surrounding the cells, and chemical analysis of the cell wallsshowed that galactose and rhamnose together with lower amounts ofglucose and mannose were present in addition to pseudomurein (Kandler &König, 1978; Kandler & König, 1985). The cell walls are also reported tocontain high levels of phosphate, comparable to that found in bacterialcell walls containing teichoic acid (Kandler & König, 1978). M1 containshomologs of genes involved in teichoic acid production in Gram positivebacteria (Bhavsar & Brown, 2006; Weidenmaier & Peschel, 2008) (Table 9,below), suggesting the presence of as-yet unidentified cell wallglycopolymers. Additionally, several genes are predicted to be involvedin exopolysaccharide production, sialic acid biosynthesis and proteinglycosylation (Table 9, below). The genome contains a homolog of theeukaryal oligosaccharyl transferase (mru0391), a membrane proteinbelieved to be involved in glycosylating proteins translocated via theSec pathway (Yurist-Doutsch, 2008) (FIG. 12). Glycoproteins derived fromthe cell wall of M1 have been shown to be highly immunogenic in sheep.The resulting antisera agglutinated M1 cells and significantly reducedtheir ability to grow and produce methane in vitro (Wedlock et al., inpreparation). Overall, polysaccharides and glycosylated molecules are amajor component of the M1 cell envelope, and their accessibility at thecell surface make these polymers viable methane mitigation targets.

Genomes of human gut methanogens encode large surface proteins that havefeatures similar to bacterial (Fricke et al., 2006; Samuel et al., 2007)adhesins. Similarly, M1 has an array of large adhesin-like proteins,much greater in number than those reported from other gut methanogens(Table 1, below). In the co-culturing experiments described above, sixM1 adhesin-like proteins were upregulated (Table 5, below), andmicroscopic examination showed co-aggregation of M1 and B.proteoclasticus cells (FIG. 9). In addition, immune sera produced bysmall peptides synthesized to correspond to four M1 adhesin-likeproteins were shown to bind specifically to immobilized M1 cells (FIG.17). Identifying highly conserved methanogen-specific features of theseadhesin-like proteins may present a pathway to vaccine development.Sixty two adhesin-like proteins are predicted to be extracellular andcontain a cell-anchoring domain (FIG. 12). These proteins represent asignificant component of the M1 cell envelope (Table 3). The largestgroup of these (44) contain a conserved C-terminal domain (M1-C, FIG. 4)with weak homology to a Big_1 domain (Pfam accession number PF02369)which may be involved in attachment to the cell wall, possibly byinteraction with pseudomurein or cell wall glycopolymers. Several ofthese proteins also contain a papain family cysteine protease domain(PF00112), and their role may be in the turnover of pseudomurein cellwalls.

A second group of 14 proteins is predicted to contain a C-terminaltransmembrane domain, suggesting they are anchored in the cell membrane.Curiously, the genome contains one adhesin-like protein (mru2147) with acell wall LPxTG-like sorting motif and three copies of a cell wallbinding repeat (PF01473), both of which are commonly found inGram-positive bacteria. There has only been one other report of aLPxTG-containing protein in a methanogen, the pseudomurein containingMethanopyrus kandleri (Boekhorst et al., 2005). Our analysis of the M.smithii PS genome revealed the presence of two LPxTG containing proteins(msm0173 and msm0411). Such proteins are covalently attached to the cellwall by membrane associated transpeptidases, known as sortases. Sortaseactivity has been recognised as a target for anti-infective therapy inbacteria (Maresso & Schneewind, 2008) and a sortase (mru1832) has beenidentified in the M1 genome.

Prophage

Phage exert a significant ecological impact on microbial populations inthe rumen, and have been suggested as biocontrol agents for rumenmethanogens (Klieve & Hegarty, 1999). M1 has 70 ORFs (mru0256-0325) overa 62 Kb GC-rich (39% G+C content) region of the genome that encode aprophage genome, designated φmru. Based on a functional annotation, φmruis partitioned into distinct modules encoding integration, DNAreplication, DNA packaging, phage capsid, lysis and lysogenic functions(Attwood et al., 2008). Within the lysis module, a gene encoding aputative lytic enzyme, endoisopeptidase PeiR (mru0320), was identified.Recombinant phage lytic enzymes have been used for controllingantibiotic-resistant bacterial pathogens (Hermoso et al., 2007), and amethanogen phage lytic enzyme may be a viable biocontrol option. We haveconfirmed the ability of recombinant PeiR to lyse M1 cells in pureculture (WO 09041831A1) (FIG. 8). PeiR represents a novel enzyme, as itdoes not show significant homology to any sequence currently in publicdatabases. The variety of methanogen cell wall types means a combinationof different lytic enzymes will be required for effective methanogenlysis in the rumen. However, the expression of PeiR and demonstration ofits effectiveness against a major rumen methanogen is an important steptowards this goal. PeiR will also be useful in increasing thepermeability of pseudomurein-containing cell walls of methanogens to aiddevelopment of genetic systems for performing gene knockouts to validatetargets, while the φmru phage itself might be useful as a genetic toolfor M. ruminantium. The prophage has also been disclosed in detail inU.S. 60/989,840 filed 22 Nov. 2007, and in PCT/2008/000248 filed 25 Sep.2008, which are hereby incorporated by reference herein in theirentirety.

Non-Ribosomal Peptide Synthetases

An unforeseen and novel feature of M1 is the presence of two largeproteins (mru0068 and mru0351) showing the distinctive domainarchitecture of non-ribosomal peptide synthetases (NRPS) (FIG. 7). Toour knowledge, this is the first report of NRPS genes identified in anarchaeal genome. HGT studies indicate that these genes may be bacterialin origin (Table 4, below). NRPSs produce a wide variety of smallmolecule natural products that have biotechnological applications aspeptide antibiotics, siderophores, immunosupressants or antitumor drugs(Amoutzias et al., 2008). The NRPS encoded by mru0068 is predicted toencode two modules, each containing condensation, adenylation andthiolation domains. The presence of a condensation domain in the firstmodule is often associated with NRPSs that make N-acylated peptides(Fischbach and Walsh, 2006). The second module is followed by a terminalthioesterase domain which is thought to release the peptide from thefinal thiolation domain. Mru0068 is surrounded by genes that encode twoserine phosphatases (mru0066, mru0071), an anti-sigma factor antagonist(mru0067), and a MatE efflux family protein (mru0069), which are likelyto be involved in environment sensing, regulating NRPS expression andexport of the NRP, respectively. Mru0068 displays full length proteinalignment with a putative NRPS from Syntrophomonas wolfei subsp. wolfeistrain Gottingen (FIG. 18), a Gram-positive bacterium known toparticipate in syntrophic interactions with methanogens (McInerney etal., 1979).

The second NRPS gene (mru0351) contains 4 modules and a thioesterasedomain. Downstream of mru0351 is another MatE efflux family protein(mru0352), presumably involved in NRP export. A third, smaller clusterof genes located elsewhere in the genome (mru0513-0516) appear to encodeNRPS-associated functions. This cluster includes a4′-phosphopantetheinyl transferase (mru0514) which primes NRPSs byadding a phosphopantetheinyl group to a conserved serine within thethiolation domain, an acyltransferase (mru0512) possibly involved in NRPacylation, a serine phosphatase (mru0515), an anti-sigma factorantagonist (mru0513), and an anti-sigma regulatory factorserine/threonine protein kinase (mru0516) that may function in sensingthe environment and NRPS regulation. Although the products of each NRPSare unknown, an analysis of adenylation domain amino acid sequencespredicts 10 residues (boxed, FIG. 7) which are important for substratebinding and catalysis. HGT studies indicate that these genes may bebacterial in origin (Table 4).

Thus, several genes in M1 are possibly involved in sensing theenvironment, and in the regulation and transport of the NRP (FIG. 7) andsuch genes are also present in the S. wolfei genome. Although the actualroles of these genes have not been defined conclusively, NRPs are knownto contribute to microbial growth and ecological interactions, thus mayprovide a means to manage methane emissions from livestock.

Comparative Genomics Analysis

To functionally compare M. ruminantium to other methanogens, 25 publiclyaccessible complete and draft phase genome sequences were subjected to aFunctional Genome Distribution (FGD) analysis (Altermann, submitted)(FIG. 13). Together with Methanobrevibater smithii, Methonsphaerastadtmanae, and Methanothermobacter thermoautotrophicus, M. ruminantiumformed a functional cluster. Within this cluster, a large number ofpredicted genes were found to be conserved (low e-value cutoff 1e-100,Table 8, below). The majority of these conserved genes were classifiedinto core biological categories, such as amino acid biosynthesis, cellcycle, central carbon metabolism, nucleic acid metabolism, protein fateand synthesis and purine and pyrimidine biosynthesis. Similarly, genesinvolved in energy metabolism, especially in the methanogenesis pathwaywere commonly shared within this functional cluster.

Most notably, only one fourth of this gene set was found to be conservedwhen compared with other functional clusters. A similar observation wasfound for conserved genes involved in the biosynthesis of vitamins andco-factors. 23 genes were found to be highly conserved within the M.ruminantium functional cluster, whereas only two genes involved incobalamin and thimaine biosynthesis were shared with methanogens outsidethis cluster, respectively. It is also interesting to note that, apartfrom Methanopyrus kandleri, pseudomurein containing methanogens havebeen functionally grouped together in the M. ruminantium cluster. Thisis clearly reflected in the 14 highly conserved genes involved inpseudomurein and exopolysaccharide biosynthesis. Outside this functionalcluster only one gene, a glucosamine-fructose-6-phosphateaminotransferase, GlmS2, was found to be conserved at this level.

While conserved gene sets cause functional clustering, strain or clusterspecific genes are responsible for differentiation. When compared to allother members of its own cluster, M. ruminantium was found to harbour468 strain specific genes (high e-value cutoff 1e-10). While the vastmajority (341 genes) was assigned to genes with hypothetical or unknownfunctional categories, a significant number of genes were identifiedassigned to relevant biological functions. Mobile elements such atransposases and prophage elements were identified as strain specific toM. ruminantium. Strain-specific genes involved in the production ofexopolysaccharides and cell surface proteins are likely to endow M.ruminantium with surface and adhesion properties distinctly differentfrom other members of the M. ruminantium functional cluster. Similarly,ten unique transport systems were identified ranging from genericmultidrug transporter to predicted pH homeostasis systems. Also 22regulatory proteins (16 involved in protein interaction and fivetranscriptional regulators) were detected. When extending the search forstrain specific genes outside its own functional cluster, the number ofidentified ORFs dropped down significantly. Only three genes coding foradhesion-like proteins and two genes involved in the production ofexopolysaccharides (a sialyltransferase and a glycosyl transferase) werefound to be M. ruminantium specific.

Comparing pseudomurein producers (PMP) to all other methanogens using arelaxed parameter set (low e-value cutoff 1e-60; high e-value cutoff1e-10; mismatch tolerance 2) revealed an interesting set of genesfunctionally specific to this subset. A number of genes involved inpseudomurein biosynthesis were identified to be functionally specific toPMPs. In addition, two genes coding for adhesion-like proteins, fourgenes predicted to be involved in exopolysaccharide production and onegene coding for a poly-gamma-glutamate biosynthesis protein, likely tobe involved in capsule synthesis, were found to be cluster specific.These genes coding for cell surface structures represent prime targetsfor vaccination based methane mitigation strategies, as their geneproducts are likely to reflect unique structures with the potential toinduce specific antibodies. Interestingly, a gene coding for theenergy-converting hydrogenase A subunit R was found to be PMP specific(including M. thermoautotrophicus and M. kandleri). This gene product isinvolved in electron transfer by reducing a ferredoxin. The Eha/Ehbcomplex is already being targeted for the development of a methanogenvaccine (FIG. 11).

Currently a bias exists for pseudomurein producing methanogen genomes.With the exception of Methanothermobacter thermoautotrophicus (isolatedfrom sewage sludge) and Methanopyrus kandleri (isolated from a submarinehydrothermal vent) all PMP methanogens were isolated either from rumen,the gastrointestinal tract or from faeces. It stands to reason thatthese closely related ecological niches facilitate common lifestyleadaption events. Such an event was detected in the presence of a highlyconserved bile salt hydrolase which is predicted to hydrolyse the amidelinkage between the bile acid carboxyl group and the glycine or taurineamino group. The presence of a bile salt hydrolase explicitly impliesthat M. ruminantium (and other rumen methanogens) is well adapted for apassage from the rumen environment through to the gastrointestinaltract. Functionally conserved oxidative stress response genes such asrubredoxin rub1 further suggest a certain tolerance of methanogens toaerobic environments and a life-cycle from oral intake, biologicalactivity in rumen and gastrointestinal tract ecologies, excretion intothe aerobic environment and a subsequent—potentially timelimited—waiting period for the next oral intake event may be proposed.How strictly anaerobic organisms such as methanogens survive exposure tohigh stress levels remains subject to further evaluation.

A recently published phylogenetic tree based on seven core enzymes(Miller, 2001) deviates in part significantly from the functionalclustering shown in FIG. 13. Anderson et al. propose three classes ofmethanogens, based on the resulting phylogenetic approximation. Incontrast, the FGD analysis revealed the presence of only two majorfunctional clusters which can each be split into further sub-clusters(FIG. 13). Most notably, Methanocorpusculum labreanum, Methanococcoidesburtonii and Methanosaeta thermophila form a distinct functional clusterunder the FGD analysis (FIG. 13, sub-cluster 1.3) but are separated intoClass II and Class III, respectively, based on the phylogeneticanalysis. These differences clearly highlight the importance of wholegenome analyses to address lifestyle adaptation processes (as describedabove) and genomic plasticity within a biotechnological context.

To investigate the broader phylogenetic placement of M. ruminantium andassess the potential level of horizontal gene transfer between archaeaand archaea and eubacteria, a Blast Heat Map analysis was performedbased on the non-redundant amino-acid database (FIG. 16). Significantheat flares were detected within archaea for the genera ofMethanococcus, and, to lesser degrees for Methanosarcina andThermococcus (FIG. 16A). Surprisingly, considerable levels of highsequence similarities were detected for the eubacterial genera ofClostridium and Bacillus, commonly found in ecological niches inhabitedby M. ruminantium. These heat flares infer a profound level of sharedand functionally similar gene sets and fortify the notion that geneticexchange between microbial domains is a common event, likely driven bylifestyle adaption forces. Analysing the relative quality of sequencesimilarity levels revealed a core set of genes commonly shared amongphylogenetically diverse methanogenic archaea (FIG. 16B). Interestingly,individual heat flares of other archaea such as Halobacteria sharinghighly conserved gene sets to M. ruminantium appear similar in shapethan flares observed for Clostridium and Bacillus, further strengtheningthe model of genetic exchange between domains. Interestingly, a moredetailed analysis of the M. ruminantium M1 ORFeome to bacterialsequences highlighted a series of gene sets not commonly found in otherMethanobacteriales such as genes involved in biotin and cobalaminbiosynthesis (Table 8, below).

Based on these observations we conducted a differential Blast Analysis(DBA) using the non-redundant database and the 26 methanogen genomes asreferences. While a DBA analysis is more liberal than FGD, it is able tohighlight gene products present in at least one methanogen genomecomprising dbMethano but not present in any other organism (nr database)and vice versa. Therefore, gene sets found to be present in methanogensbut not in other microbes might represent prime targets for methanemitigation strategies. Using a minimum DBA value of four, 117 genetargets were identified to be conserved between M. ruminantium andmethanogens but not present in other organisms. Notably, 21 genespredicted to be associated to the cell envelope were identified,including cell surface proteins and genes involved in exopolysaccharideand pseudomurein biosynthesis. One gene coding for a polysaccharidebiosynthesis protein and two adhesion-like proteins were among the mostprominent targets (DBA value of −6). All three genes are involved incell-cell interaction and could represent targets for methanogenmodulation. Interestingly, a single adhesion like protein was identifiednear the origin of DNA replication, conserved between M. ruminantium andother organisms but absent in methanogens (FIG. 10).

Closest hits were found to Coprococcus eutactus (Acc: ZP_02205388.1,isolated from human faeces), Streptococcus sanguinis (Acc:YP_001036038.1, isolated from human dental plaque), Peptostreptococcusmicros (renamed as: Parvimonas micra, Acc: ZP_02093886.1, isolated fromhuman gut), Arcanobacterium pyogenes (Acc: AA043108.1, commonly found onmucosal surfaces of livestock) and Bacillus weihenstephanensis (Acc:YP_001647931.1, human pathogen). All of those organisms are able tointeract with either animal or human hosts and it is tempting tospeculate that this adhesion reflects a specific lifestyle adaptation ofM. ruminantium to the rumen and, possibly, to the gastrointestinal tractenvironment. This clearly strengthens the previous hypothesis modelledon the Blast Heat Map proposing a significant level of genetic exchangebetween M. ruminantium and certain genera of Eubacteria. Similarly, twoadjacent glycosylhydrolases of the GT2 family were found to be shared ina similar way. For both proteins, top Blast hits link to Clostridia andBacilli while their function was associated to the transfer of sugarunits to teichoic acids which in turn are covalently linked to the cellwall thus providing M. ruminantium with a methanogen-unique outercell-surface structure.

Genes predicted to be involved in hydrogen metabolism and methanogenesissuch as the energy-converting hydrogenase B (Ehb) and the tungstenformylmethanofuran dehydrogenase (Fwd) were found to be methanogenspecific. Also, another gene involved core functions, a Fibrillarin-likearchaeal protein, commonly thought to participate in processingpre-ribosomal rRNA, was found to be methanogen specific. This isinteresting, as only methanogen genera were excluded from thenon-redundant database, but not other archaea. Therefore, this genemight present an opportunity to target methanogens at an essentialfunction. An in-depth analysis of the presence/absence of this gene inthe methanogen genomes revealed an interesting distribution pattern.With the exception of Methanospirillum hungatei members of functionalcluster 4 (FIG. 13) do not harbours this gene, while cluster 3predominantly features this archaeal gene, with the notable exception ofMethanococcoides burtonii, Methanosaeta thermophila and Methanosphaerastadtmanae. It might be noteworthy, that within methanogens this gene iseither absent or present, creating a highly conserved target with lowgenetic drift. Although not universally conserved, this gene might offerthe opportunity to specifically target the majority of cluster 3methanogens, thus including those of rumen origin, for methanemitigation strategies.

Identification of Targets for Methane Mitigation

Several approaches were used to define potential gene targets from M1for CH₄ mitigation via chemogenomic and vaccine approaches (FIG. 1).Genes suitable as chemogenomics targets were identified using acombination of metabolic profiling, review of the literature pertainingto the biochemistry and physiology of methanogens, and comparativegenomics. The 33 candidate genes commonly identified by these approachesare shown in FIG. 1A. The full list of ORFs identified as chemogenomictargets by metabolic profiling of M1 and literature can be found inTable 5. Comparative studies were based on M1 and 26 complete and draftphase methanogen genome sequences, using a functional genomedistribution (FGD) analysis (Table 3, FIG. 13). This analysis of wholegenome gene conservation among methanogens showed that M1 and othermembers of the Methanobacteriales formed a functional cluster thatshared a large number of conserved genes predicted to be involved incore biological functions (low e-value cut-off 1e-100, Table 3). Inaddition, a differential blast analysis (DBA) was conducted using thenon-redundant (nr) database and a methanogen genome sequence database(dbMethano). The DBA analysis highlighted genes present in at least onemethanogen genome within dbMethano but not present in any other organismwithin the nr database and vice versa (FIG. 10), thus identifyingmethanogen-specific genes. The majority of the 33 selected conserved andmethanogen-specific genes encode enzymes that fall within the energymetabolism category, mainly within the methanogenesis pathway (Table 9).This also included several methanogenesis marker proteins found inmethanogen genomes, but currently without defined function. Most ofthese methanogenesis enzymes are located within the cell cytoplasm, andtherefore have been tagged as key targets for inhibitor discovery via achemogenomics approach (FIG. 11).

The alternative approach of inducing the ruminant immune system toproduce salivary antibodies against conserved features of rumenmethanogens is an attractive methane mitigation strategy. The rumenepithelium is not immunologically active, and rumen contents do notcontain complement proteins, therefore specific immune responses in therumen do not occur. The effectiveness of a vaccination approach relieson the binding of salivary antibodies to methanogen surface featureswhich results in their inactivation or clearance from the rumen.Vaccines are typically composed of proteins or polysaccharides derivedfrom killed or attenuated whole cells or components presented on theoutside of the cell such as flagella, capsules, cell walls, fimbrae, orsecreted toxins. In the case of rumen methanogens, the primary vaccinetargets are likely to be surface-exposed or membrane-associated proteinsthat are conserved among methanogens or archaeal species and whichencode functions vital to methanogen growth and survival in the rumen.In silico analysis of the M1 ORFeome (all ORFs) identified an initialpool of 572 ORFs containing one or more transmembrane helices (TMH) orsignal peptide (SP) indicating a cell membrane or cell surface locationand therefore potential vaccine targets. Those ORFs with a top BLAST hitto a non-methanogen or with no homology to the nr database were removedfrom the analysis, as were transposase sequences (which are unlikely torepresent good vaccine targets), while adhesin-like ORFs are dealt withseparately above. This gave a new total of 337 ORFs. Examination of theremaining 337 ORFs, assessing their predicted function, degree ofconservation among methanogens and the nature of their transmembranestructures, refined the list to 71 ORFs (FIG. 1B). Heterologousexpression of membrane proteins with more than 4 TMHs has been difficultin RV studies of other microbes (Vivona et al., 2008). Therefore, acut-off of 4 THMs was applied to define two final groups: Group A with47 ORFs with 4 or fewer TMHs suitable for cloning and heterologousexpression studies; and Group B composed of 24 ORFs with more than 4TMHs more suitable for a synthetic peptide-directed vaccine approach.

The majority of vaccine targets identified above correspond tohypothetical proteins of unknown function. While these ORFs are presumedto be of value to M1, their importance to M1 growth and survival in therumen is not evident, and therefore they are of lower priority asvaccine candidates. Of the remaining ORFs, those involved in energymetabolism are again prime vaccine candidates (FIG. 11). Of particularinterest is the Mtr enzyme complex, which catalyses the essentialmethanogen function of transferring the methyl group from methyl-H₄MPTto CoM, coupled to the efflux of Na⁺ ions (Lienard et al., 1996). Threeof the Mtr subunits (MtrEDC) are each predicted to have >4membrane-spanning regions and, in each of the membrane-spanning regions,the transmembrane helices have peptide loops located outside the cellmembrane. These loops are potential antibody binding sites. Wesynthesised peptides corresponding to the loop regions of MtrE, MtrD andMtrC which were coupled to a carrier protein and then used as antigensto vaccinate sheep. The resulting immune sera bound specifically toimmobilized M1 cells (FIG. 17), demonstrating the feasibility of such apeptide-directed RV approach.

Vaccine Target Identification Results

TMHMM predicted 542 ORFs to contain one or more transmembrane (TM)domains, 243 of which are also predicted to contain a signal peptide(SP). A further 30 ORFs were predicted to contain a signal peptide butno TM domain. This gave a total pool of 572 ORFs as potential vaccinecandidates for the M. ruminantium M1 genome. Blast analyses revealedORFs that had a top blast hit to a non-methanogen or had no homology tothe NR database. These ORFs were removed from the analysis at this pointas were transposase sequences (which are unlikely to represent a goodvaccine target) and adhesin-like ORFs which were dealt with separately.This gave a new total of 339 ORFs. Those genes which are presently onlyfound in M. ruminantium M1 and provide important information about rumenmethanogens.

Methanogens are strict anaerobes and are thus present challenges forculturing in vitro and genetic techniques for validating both drug andvaccine targets particularly the Methanobacteria with their thick cellwalls are still being established. As such, a further manual analysis ofthe 339 ORFs functions was undertaken re-examining their functionalannotation, their conservation among methanogens and the prediction oftheir transmembrane structures and subcellular location which refinedthe target list to 71 ORFs which are presently specific to methanogensor archaea. Because expression can be challenging for membrane proteins,a cut-off of four TM domains was applied to expression studies of M.ruminantium M1 (multiple TMs can be indicative of a surface exposedprotein, but conversely, they are known to impair heterologousexpression in Escherichia coli) This resulted in a final two groups;Group A—47 vaccine targets (less than or equal to 4 TM) suitable forfurther cloning and expression studies and Group B—24 vaccine targets(greater than 4 TM) more suitable for a synthetic peptide directedvaccine approach. The adhesin-like ORFs were treated as a separatevaccine target list. Vaccine targets have also be disclosed in detail inU.S. 60/989,841 filed 22 Nov. 2007, and in PCT/2008/000249 filed 25 Sep.2008, which are hereby incorporated by reference herein in theirentirety.

Example 3: Overview Genome Sequencing

Methanobrevibacter ruminantium was chosen for genome sequencing becauseof its prevalence in the rumen under a variety of dietary conditions(based on cultivation and molecular detection data), the availability ofcultures, its amenity to routine growth in the laboratory, and therelatively large amount of previous studies and background literatureavailable for this organism. A significant number of the genes withinthe M. ruminantium have been assigned a function, and have therebyallowed a detailed picture of this organism's lifestyle within therumen. M. ruminantium's dependence on simple substrates (H₂+CO₂,formate) and its interaction with the rumen environment via surfaceproteins and exopolysaccharides are important targets for inhibition.Similarly, the SPIDRs hold promise for both specific targeting of M.ruminantium and for future genetic manipulations to assist indetermining gene function. The sequence data elucidates the metabolismof this organism and how it interacts with other microbes, and points toconserved systems and components among methanogens that can beinactivated to prevent or reduce methane formation in the rumen.

Drug Discovery for Reducing Rumen Methane Emissions

The completion of the M. ruminantium genome now enables the full powerof ‘-omic’ approaches to be used for developing novel drugs for reducingmethane emissions. A rational drug discovery approach to the developmentof effective methane mitigation agents, analogous to the establishedapproaches utilised for developing novel antibiotics against humanpathogens, should proceed through several stages including: targetidentification; target/pathway validation; lead identification;verification of effectiveness of lead in rumen-like conditions; andultimately, testing in animal trials (Gerdes et al. 2006; Pucci 2006;Galperin 2007). A priori, it is important to know the breadth ofmethanogen diversity in the rumen, and this has recently been summarisedin a meta-analysis incorporating data from 14 phylogenetic studies(Janssen and Kirs, 2008). The study has shown that the vast majority ofrumen archaea (92.3%) fall into three clades, the generaMethanobrevibacter (61.6%) and Methanomicrobium (14.9%) and anuncultured group termed rumen cluster C (15.8%) of as yet unknownphysiology (Janssen and Kirs, 2008). Methanogens typically only accountfor approximately 1-4% of the total microbial community (Janssen andKirs 2008).

Inter-genome comparisons of representative organisms, including relevantgut methanogens (e.g., Methanobrevibacter smithii, Methanospirillimhungatei and Methanosphaera stadtmanae), other rumen microorganisms(bacterial, fungal and prozoal) and mammals can aid in theidentification of suitable targets (Samuel et al. 2007; Fricke et al.2006). Overall, rumen methanogens should be somewhat easier to inhibitthan bacteria, fungi or protozoa given that they are the only residentarchaea in the rumen and are well known to possess several unique traitsincluding distinctive cofactors, cell wall chemistries and lipids.Furthermore, they tend to have smaller genomes with less metaboliccapability, tend to be less adaptable with fewer regulatory systems andare probably less able to develop resistance to drugs.

Given the diversity of methanogens in the rumen, the aim of developing afull spectrum anti-methanogen ‘magic bullet’ for complete mitigation ofemissions would necessitate the targeting of enzymes that are essentialto all rumen methanogens under normal rumen growth conditions. However,partial inhibition may also be desirable for extended periods of time,due to the potential decrease in the efficiency of feed conversionresulting from feedback inhibition of ruminal fermentation (Hegarty1999; Russell and Rychlik, 2005). Significant partial reductions inmethane emissions which might be more sustainable could also possibly beachievable by limiting the targeting to specific phylogenetic groups,such as the dominant Methanobrevibacteria. Methanogens grow slowlycompared to rumen bacteria and are prone to being flushed out of therumen (Janssen and Kirs 2008).

Analysis of the M. ruminantium genome, combined with the genomecomparison and a consideration of the literature that has identifiedarchaeal/methanogen-specific enzymes or has demonstrated theessentiality of enzymes/pathways has allowed us to generate a list oftargets of interest (Tables 2 and 3, below). General targets areasinclude the methanogenesis pathway, energy metabolism, transcription,protein synthesis, cell wall synthesis, lipid synthesis, cofactorsynthesis, and some key central carbon metabolic enzymes that areimportant links between essential pathways. Target prioritisation forintroducing enzymes into the work stream is based on the ultimate aim ofobtaining enzymes for high-throughput screening and crystal structuredeterminations for in silico lead identification. Targets are spreadover multiple susceptible pathways to minimise risk. Prioritisationtakes into consideration the presumed essentiality of target, theexpected ease of expression of the proteins (e.g., size, number ofsubunits and presence of transmembrane domains), availability of assays,expected ‘druggability’ and availability crystal structures ofhomologous enzymes (Pucci, 2006; Hopkins and Groom, 2002).

There are several factors relevant to the development of future smallmolecule inhibitors of methanogens. These include that they should haveminimal toxicity to the host animals, minimal accumulation or toxicityin any downstream products for human consumption, minimal deleteriouseffects on beneficial microorganisms responsible for normal fermentationin the rumen, and minimal downstream environmental impact. Ideally, theyshould also be inexpensive, given the current cost of carbon, beimpervious to the large hydrolytic capacity of the rumen and should havereduced potential for allowing resistance to develop amongst the rumenmethanogens. In the best circumstances, the concentration of inhibitorrequired should be low enough to prevent any rumen microbes fromutilising the inhibitor as a substrate for growth and minimise overallcosts (Weimer, 1998). It would be very beneficial if futureanti-methanogen compounds that satisfy the above criteria can also helpto reduce methane emissions emanating from other sources such as ricepaddies. The goal of providing long term reductions in rumen methaneemissions over decades will likely require a suite of non over-lappinganti-methanogen compounds. Compounds that are shown to inhibit an enzymein an in vitro high-throughput assay should sequentially be verified fortheir effectiveness in pure culture experiments, followed by in vitrorumen simulations, and finally in short term and long term animaltrials.

In the last decade there has been growing concern, as evidenced byrecent EU legislation banning their use (McAllister and Newbold, 2008),about the use of antibiotics and other feed additives as growthpromoters largely due to the development of antibiotic resistance.Consequently, even though methanogens are usually not thought to bedirectly implicated as a causative agent in pathogenic disease (Macarioand Macario, 2008), it may be wise to avoid over-reliance onanti-methanogen agents that seek to inhibit enzymes that are commonbetween them such as those in pseudomurein and peptidoglycan synthesis.Significantly, approximately two-thirds of antibiotics in use todayagainst pathogenic microorganisms target cell wall synthesis, many ofthese the transpeptidation reaction catalysing the closure of bacterialpeptidoglycan, but enzymes that catalyse earlier steps are beingincreasingly investigated as alternatives (Hammes et al. 1979). One ofthe main reasons for this is that the final steps of cell wall synthesisoccurs extracellularly and are therefore the drugs are less prone toinactivation.

The recent development of an anti-Mycobacterium tuberculosis drug thattargets the E subunit of the F₀F₁-ATPase that is also present in humanshighlights the fact that even relatively small differences in structurecould possibly be exploited to discover novel anti-methanogen compounds(Andries et al. 2005). The implication of this finding is that manyessential methanogen enzymes that are not methanogen-specific. Targetsthat share <30-40% identity with their bacterial or eukaryalcounterparts could ultimately be exploited for designing effectiveinhibitors.

The number of enzymes or pathways in methanogens demonstrated to beessential to help guide prioritisation of targets is relatively limited.Historically, the methanogenesis pathway itself has been targeted innumerous experiments using halogenated compounds such as chloroform thatinhibit the terminal step of methanogenesis catalysed by methyl coenzymeM reductase, but these are not sustainable due to toxicity andenvironmental concerns (hypertext transferprotocol://www.maf.govt.nz/mafnet/rural-nz/sustainable-resource-use/climate/green-house-gas-migration/ghg-mitigation-05.htm).In only a couple of other cases has a methanogen enzyme inhibitor beenused to check whether rumen methanogen isolates hare also inhibited inpure culture, for example the targeting of the RFA-P synthase (Dumitruet al. 2003) and HMG CoA reductase with statins (Miller and Wolin,2001). In addition, some additional understanding of growthcharacteristics of methanogens in the rumen may be needed. For example,researchers are still determining the extent to which rumen methanogensutilise amino acids in the rumen, which vitamins are used or enhancegrowth or the degree, if any, to utilise purines and pyrimidines. Thusat the moment, it may less desirable to target enzymes dedicated to thesynthesis of amino acids, purines or pyrimidines.

The analysis of the M1 genome has provided new perspectives on thelifestyle and cellular processes of this prominent rumen methanogen. Thegenome sequence confirms the hydrogenotrophic lifestyle of M1 and geneexpression data indicate that formate may be an important substrate formethanogenesis during syntrophic interaction with B. proteoclasticus.The ability of short chain alcohols to stimulate growth on H₂ but notsupport growth themselves is intriguing. We speculate that methanol orethanol are oxidised by the NADP-dependent alcohol dehydrogenases andthe reducing potential used to form F₄₂₀H₂ using NADPH-dependent F₄₂₀dehydrogenase, thus augmenting the cellular pool of F₄₂₀H₂. Thismetabolism of alcohols could spare some of the H₂ or formate normallyused to produce F₄₂₀H₂ and would explain the stimulation of growth byalcohols in the presence of H₂. The lack of a means of reducingferredoxins with electrons from alcohols would explain why growth is notpossible on alcohols alone. Further work will be required to test thishypothesis.

The abundance of genes encoding adhesin-like proteins in M1 indicates asignificant ability to modulate cell surface topology. While the exactrole of these proteins is currently unknown, initial observations fromco-culture experiments indicate that at least some are involved inmediating close associations with hydrogen-producing bacteria in therumen and others may be concerned with similar interactions with rumenprotozoa and fungi.

The φmru prophage sequence within the M1 genome yielded the PeiR enzymewhich is able to lyse methanogen cells. The variety of methanogen cellwall types means a combination of different lytic enzymes would berequired for effective methanogen lysis in the rumen. However, theexpression of PeiR and demonstration of its effectiveness against amajor rumen methanogen is an important step towards this goal. The PeiRenzyme and the φmru phage may also be useful in increasing thepermeability of M1 and other pseudomurein-containing methanogens tofacilitate DNA entry and for developing tools for genetic manipulationof M1.

Methanogens are not known as producers of secondary metabolites, so thediscovery of two NRPS genes was surprising, and to our knowledge, theyare the first reported in an archaeal genome. Non-ribosomal peptides(NRPs) are known to contribute to microbial growth and ecologicalinteractions and therefore their function is of interest as they couldlead to a means of modulating methanogen growth.

The metabolic profiling and comparative genomics carried out in thisstudy identified several sets of conserved, methanogen-specific genesthat are currently being investigated further in our laboratory.Chemogenomic targets are being investigated via heterologous expressionof genes in Escherichia coli coupled with the development of bioassaysfor screening these enzymes against libraries of chemical compounds tofind specific inhibitors with efficacy at low concentrations. Vaccinecandidate proteins with <4 TMHs are being investigated via heterologousexpression in E. coli and vaccination of sheep. We have also shown theuse of synthetic peptides in a reverse vaccinology approach to elicitspecific antibody responses against M1 proteins with >4 TMHs. Thisdemonstrates that membrane-embedded M1 proteins, that are unlikely to beamenable to expression in a heterologous host, are viable targets asvaccine antigens.

A wider representation of rumen methanogen genomes will be essential toverify that the selected vaccine and chemogenomics targets are conservedamong other rumen methanogens, and ensure a successful, long-term CH₄mitigation technology for the rumen. The wealth of biologicalinformation provided by the M1 genome represents a significantadvancement for ruminant methane mitigation efforts, aimed atidentifying anti-methanogen technologies with broad efficacy.

Example 4: A₁A_(o) ATP Synthase Cloning and Characterisation

We designed and developed an over-expression system for theA₁A_(O)-ATPase from Methanobrevibacter ruminantium andMethanobrevibacter smithii to allow the subsequent purification andcharacterisation of the A₁A_(O)-ATPase.

Methanobrevibacter ruminantium and Methanobrevibacter smithii PCRCloning and Introduction of Hexa-His Tag by PCR Overlap Extension

To study the biochemical properties of the M. ruminantium A₁-ATPase, weprepared inverted membrane vesicles and tested for ATP hydrolysisactivity. We were unable to detect any significant levels of ATPhydrolysis activity from inverted membrane vesicles of M. ruminantium.Due to the limited amount of cells that can be prepared at any giventime for M. ruminantium, we undertook a heterologous over-expressionapproach to produce the A₁-ATPase. For this, we cloned the M.ruminantium A₁-ATPase genes as a 6.3 kb BamH1-Xba1 PCR product into theexpression vector pTrc99a, generating plasmid pTrMbrA1. To insert theHIS-tag at the N-terminal of Subunit-A PCR overlap extension wasconducted. Using this approach, we generated a clone named pTrMbrA1HISwhich contains the genes encoding for the M. ruminantium A₁-ATPase inthe E. coli expression vector pTrc99a, and introduced a Hexa-Histidinetag onto the N-terminal of Subunit A. We also cloned the A_(o) geneswith the A₁ genes to construct a full-length A₁A_(o) ATP synthaseexpression plasmid. This was named pTrMbbrA₁A_(o)His9. In addition, wecloned the A₁-ATPase of Methanobrevibacter smithii as a 6.3 kb PCRproduct into the E. coli expression vector pTrc99a generating theplasmid pTrMbsA1. To facilitate purification, we introduced aHexa-Histidine tag onto the N-terminal of the M. smithii Subunit-A byPCR overlap extension. This was named pTrMbsA1 HIS. The lists of primersand plasmids from this study are shown below.

Primers

SEQ ID Primer Name Primer Modifications NO: MbrA1FWD AAATTT GGATCCGGAATCTTAGGTTAGGAG (BamHI) 7590 GTCAAT MbrA1REV AAATTT TCTAGATAACAAGCAAAATATGAAT (XbaI) 7591 TGC MbrA1HisFWDATGCATCATCATCATCATCATAGAGGAACT HEXA-HIS TAG 7592 CAAATGTATGAAMbrA1HisREV ATGATGATGATGATGATGCATCCCATCTGC HEXA-HIS TAG 7593GACGATAACAGG MbrA1His_MID TTAGACAAGTTCTTA GTCGAC TCTG (Sal) 7594MbrAOREV AGAGACAATTTTATCTGCCCCA GAGCTC AT (Sac) 7595 MbrA1FWDMbrAATTTAATT ACCATG GTGATTTATTATGGCAA (Nco) 7596 1ASeq1 TTGCAGGTCCTGTTATCGTC7597 MbrA1ASeq2 GGACATTCCACTTATTACCGC 7598 MbrA1ASeq3ACTTATCCGAACCGGTTACTC 7599 MbsA1FWD AAATTTTAA GGATCC AATCTGTATGAGCTCABamHI 7600 G MbsA1REV AAATTTGTCGACCAATTACACAAAAAGATGA SalI 7601 GCCGTTACMbsA1HisFWD ATGATTCATCATCATCATCATCATATCGAAG HEXA-HIS TAG 7602GAAAAATTATTAAA MbsA1HisREV ATGATGATGATGATGATGAATCATTTAACCA HEXA-HIS TAG7603 TCTCTACCCCAATA MbsA1_AatIIREV ATTTATCCACATATG GACGTC CTTTCCTTA(AatII) 7604 MbsA1Seq1 CCTCTGAAGGATCATCTGAT 7605 MbsA1Seq2AGCATTGCTTCTGAAGGTGAA 7606 MbsA1Seq3 GAGTAAACACTATTGGTACTA 7607

Plasmids

Plasmid Name Details Features pTrMbrA1 Methanobrevibacter ruminantiumA₁-ATPase cloned into expression vector pTrc99a as a 6.3 kb Bam/Xbafragment pTrMbrA1HIS Methanobrevibacter ruminantium A₁-ATPase Hexa-cloned into expression vector pTrc99a as a His Tag 6.3 kb Bam/Xbafragment. N-terminal Hexa- Histidine tag on Subunit A pTrMbrA1AOMethanobrevibacter ruminantium A₁A_(O)- Hexa- ATPase cloned intoexpression vector His Tag pTrc99a as a Nco/Xba fragment. N-terminalHexa-Histidine tag on Subunit A pTrMbsA1 Methanobrevibacter smithiiA₁-ATPase cloned into expression vector pTrc99a as a 6.3 kb Bam/Salfragment pTrMbsA1HIS Methanobrevibacter smithii A₁-ATPase Hexa- clonedinto expression vector pTrc99a as a His Tag 6.3 kb Bam/Sal fragment.N-terminal Hexa- Histidine tag on Subunit AOver-Expression and Purification of pTrMbrA1HIS and pTrMbsA1 HIS asAnalysed by Western Blotting

As noted above, we generated a clone named pTrMbrA1HIS which containsthe genes encoding for the M. ruminantium A₁-ATPase in the E. coliexpression vector pTrc99a, and introduced a Hexa-Histidine tag onto theN-terminal of Subunit A. We expressed the plasmid pTrMbrA1HIS in the E.coli strain DK8, and purified the expressed protein complex byNi-affinity chromatography. We were able to detect Subunit-A via bothWestern and MALDI-TOF/TOF analysis. Subunit-A was found to be running atan incorrect molecular mass of approximately 24 kDa compared to the 65kDa predicted molecular mass. We found that this discrepancy was causedby a mutation within Subunit-A. This mutation has now been corrected,and we have repeated the over-expression and purification with the newconstruct. Furthermore, we have also created a clone to express the fulllength A₁A_(O)-ATPase in E. coli. The purification and characterisationof the A₁A_(O)-ATPase from M. ruminantium has proceeded accordingly.

From Western analysis of the M. smithii over-expression, two dominantbands can be observed. One protein band runs above the 72 kDa marker,and the other runs at approximately 33 kDa. The expected size of the M.smithii Subunit-A is 64.8 kDa, therefore the protein band running atapproximately 72 kDa is likely the Mbb. smithii Subunit-A, and the lowerband running at 33 kDa is likely a breakdown product, or unassembledSubunit-A which still retains the HIS-tag. We are proceeding to furtherpurify the Ni-affinity eluted fractions, through eitherPEG-fractionation or gel filtration with the aim to remove the lowercontaminating band, while still retaining A₁-ATPase activity. We haveassayed the ATPase activity of the eluted M. smithii A₁-ATPase and foundthe sample to hydrolyse with a specific activity of approximately 0.4Units/mg.

Mbb. Ruminantium A₁A_(o) ATP Synthase Expression in a Foreign Host

The Mbb. ruminantium A₁A_(O)-ATPase was expressed in E. coli strains DK8(Δatp), BL21 and C41 (FIG. 24A-D). All strains showed a decrease ingrowth after induction of expression with 1 mM IPTG. BL21 showed thebest growth (FIG. 24A) and expression of the A₁A_(O)-ATP synthase andtherefore was chosen as a suitable expression host for subsequentpurification. Growth of the induced cultures (BL21, C41 orDK8/pTrMbbrA₁A_(o)His9) is at a reduced rate compared to the non-inducedcontrol culture in all 3 strains (see FIG. 24A-C), a phenomenon that isa good indication of foreign protein over-expression in E. coli. Toexamine the localization of the recombinant A₁A_(O)-ATP synthase in E.coli the cell debris, cytoplasm and membranes were examined by SDS-PAGEand immunoblotting. Purified F₁F_(o)-ATPase (his-tag on 3-subunit) fromTA2.A1 enzyme was used as a positive control (FIG. 24D). The enzyme waslocalized in the membranes and not in the cytoplasm indicating thepresence of a properly assembled enzyme. pTrMbbrA₁A_(o)His9 was able tobe expressed in E. coli strains BL21, C41 or DK8, with the best growthand overexpression in BL21 (data not shown). These results also show theA₁A_(O)-ATP synthase is specifically localizing to the membranepreparation in both BL21 and DK8 cells.

Extraction of the A₁A_(O)-ATP Synthase from Cell Membranes

The Mbb. ruminantium A₁A_(O)-ATPase was expressed in the E. coli DK8(Δatp) and BL21 (FIG. 24A-D). Previously, we were able to solubilize theMbb. ruminantium A₁A_(o) using 2% DDM, and we are able to semi-purifythe Mbb. ruminantium A₁A_(o) ATP synthase by exploiting the introducedhexa-histidine tag on the A subunit (elution at 120 mM imidazole, seeFIG. 25). However, solubilisation was considered limited with only about40% of the tagged protein being extracted from membranes. Therefore, todetermine the detergent with the optimal solubilisation effect, E. coliBL21 inverted membrane vesicles were diluted in solubilsation buffersupplemented with different detergents to concentrations of 0.5, 1,2 or4%, and a concentration of 5 mg protein/ml. Solubilisation was performedunder gentle stirring overnight at 4° C. or at room temperature with DM,DDM, Triton X-100, Cymal-6, CHAPS, cholate, octylglucoside andfos-choline. SDS was used as a positive control as it solubilized 100%of the membranes. The soluble and insoluble fractions were analyzed bySDS-PAGE and immunoblotting. Comparison of the immunoblots revealed thatfos-choline had the best solubilisation effect (90-100%), followed byDDM (40%), DM (35%) and cymal-6 (39%). Triton X-100 and octylglucosidewere weak (>20%). CHAPS and cholate led to a significant degradation ofthe enzyme (a smear) and were therefore not feasible. However, afteractivity was measured of the solubilized membrane protein, the bestdetergent was DDM or cymal-6 both liberating 40% of the ATPase andmaintaining activity the highest activity. The Fos-choline samples didnot contain enzyme activity regardless of concentration.

Purification of the Mbb. ruminantium A₁A_(O)-ATPase

BL21 containing pTrMbbrA₁A_(o)His9 induced with 1 mM IPTG and expressionconducted for 4 hours at 37° C. Membranes were prepared by French-press,and solubilized with 2% DDM at 4° C. overnight in the presence of 0.1%TCEP (a reductant). The purification was then routinely performed byIMAC and the bound protein subjected to 10, 20, 40 then 60 mM imidazolewash steps before elution at 100 mM imidazole. It should be noted thatelution of a significant amount of A₁A_(o) protein is observed at 60 mMimidazole, however to ensure a very clean preparation this step wasessential to remove contaminating proteins. To remove additionalcontaminants, the eluant was PEG-precipitated for 1 h at roomtemperature with 10% PEG₆₀₀₀ followed by 15% PEG₆₀₀₀. The first stepprecipitation removed contaminating proteins (fraction split), thesecond precipitates the A₁A_(o) ATPase.

The purified Mbb. ruminantium A₁A_(O)-ATPase contained all 9 subunits,which was confirmed by MS/MS (FIGS. 25A and B). The K-subunit appearsboth as a monomer and as an oligomer on a 14% SDS-PAGE gel. When thisenzyme preparation was TCA-treated, the oligomers were no longer seen,and a strong band of the K monomer was observed. This observation isindicative of an SDS-stable K ring. This was further confirmed byisolation of monomers of the K-subunit by methanol/chloroform extraction(FIG. 27). This K ring preparation is being used for antibody trials.

Purified Mbb. ruminantium A₁A_(o) ATP Synthase Characterization

Two enzyme preparations were studied. Purified ATP synthase from BL21and recombinant enzyme expressed in E. coli DK8 membranes. PurifiedA₁A_(o) ATP synthase was examined for ATPase activity using theinorganic phosphate assay. After examining current literature on theMethanosarcina mazei and Methanococcus jannaschii A₁A_(o) ATP synthases,it was decided to examine activity at 39° C. and a pH value of 6.5 in abuffering system that mimics the Na⁺ concentration in the rumen (70-137mM Na⁺).

To determine the kinetics of ATP hydrolysis, the reaction was started at39° C. by addition of Na₂-ATP to a final concentration of 2.5 mM. 16 μgof protein was used in each end-point assay. Background ATPase activitygenerated by thermal hydrolysis of ATP or contaminant ATP in buffer orenzyme was subtracted (these totalled <5% of the final value shown; FIG.29A). The influence of Mg²⁺ on the kinetics of ATP hydrolysis was alsoevaluated. The reaction was started by addition of Tris-ATP to a finalconcentration of 2.5 mM. 16 μg of protein was used in each end-pointassay. Background ATPase activity generated by thermal hydrolysis of ATPor contaminant ATP in buffer or enzyme was subtracted (these totalled<5% of the final value shown; FIG. 29B).

ATPase activity was tested over a pH value range from 5.5 to 8.5 and inpresence and absence of Na⁺ using the purified recombinantA₁A_(O)-ATPase. The reaction was started at 39° C. by addition ofNa₂-ATP or Tris-ATP to a final concentration of 2.5 mM. 16 μg of proteinwas used in each end-point assay. Background ATPase activity generatedby thermal hydrolysis of ATP or contaminant ATP in buffer or enzyme wassubtracted (these totalled <5% of the final value shown; FIG. 29C). Thestability of the purified and membrane-bound (in DK8 membranes) Mbb.ruminantium A₁A_(o) ATP synthase was also tested. ATPase activity wasexamined each day after the preparation of either purified recombinantor DK8 membrane bound Mbb. ruminantium A₁A_(o) ATP synthase. Thereaction was started at 39° C. by addition of Na₂-ATP to a finalconcentration of 2.5 mM. 16 μg of purified protein or 0.5 mg invertedmembrane vesicles was used in each end-point assay. Background ATPaseactivity generated by thermal hydrolysis of ATP or contaminant ATP inbuffer or enzyme was subtracted (these totalled <5% of the final valueshown; FIG. 29D).

To gain insight to whether the purified or membrane-bound Mbb.ruminantium A₁A_(o) ATP synthase is functionally coupled to an ionicdriving force (H⁺ or Na⁺), tributylin (TBT) was tested as an inhibitor.TBT is a well characterized F_(o) and A_(O) channel inhibitor of ATPsynthases. To examine the coupling ion used by the Mbb. ruminantiumA₁A_(o) ATP synthase, the effect of amiloride, a known Na⁺-coupledV-type ATPase and Na⁺ channel inhibitor was also examined.

The effects of TBT and DCCD on ATPase activity were investigated asfollows. E. coli DK8 (Δatp) inverted membranes containing therecombinant A₁A_(O)-ATPase were used to determine the effects of theinhibitors TBT (200 μM) and DCCD (250 μM) at different pH values. ATPaseactivity was measured in presence of 130 mM Na⁺ (FIG. 30A) and inabsence of Na⁺ (FIG. 30B). After preincubation with the inhibitor for 20min at room temperature (TBT or DCCD), the reaction was started at 39°C. by addition of Na₂-ATP or Tris-ATP to a final concentration of 2.5mM. 0.5 mg inverted membrane vesicles was used in each end-point assay.Background ATPase activity generated by thermal hydrolysis of ATP orcontaminant ATP in buffer or enzyme has been subtracted (these totalled<5% of the final value shown; FIGS. 30A and 30B).

Tributylin inhibition of ATP hydrolysis by purified recombinant Mbb.ruminantium A₁A_(o) ATP synthase was then evaluated. After preincubationwith the inhibitor tributylin (TBT) for 20 min at room temperature, thereaction was started at 39° C. by addition of Na₂-ATP to a finalconcentration of 2.5 mM. 16 μg of protein was used in each end-pointassay. Background ATPase activity generated by thermal hydrolysis of ATPor contaminant ATP in buffer or enzyme was subtracted (these totalled<5% of the final value shown; FIG. 30C). Amiloride inhibition of ATPhydrolysis of the Mbb. ruminantium A₁A_(o) ATP synthase was evaluatednext. After preincubation with the inhibitor tributylin (TBT) for 20 minat room temperature, the reaction was started at 39° C. by addition ofNa₂-ATP to a final concentration of 2.5 mM. 16 μg of protein was used ineach end-point assay. Background ATPase activity generated by thermalhydrolysis of ATP or contaminant ATP in buffer or enzyme was subtracted(these totalled <5% of the final value shown; FIG. 30D).

Tributylin inhibition of ATP hydrolysis by the Mbb. ruminantium A₁A_(o)ATP synthase was also tested in DK8 and native membranes. Afterpreincubation with the inhibitor tributylin (TBT) for 20 min at roomtemperature, the reaction was started at 39° C. by addition of Na₂-ATPto a final concentration of 2.5 mM. 0.5 mg inverted membrane vesicleswas used in each end-point assay. Background ATPase activity generatedby thermal hydrolysis of ATP or contaminant ATP in buffer or enzyme wassubtracted (these totalled <5% of the final value shown; FIG. 31A).Amiloride inhibition of ATP hydrolysis of purified recombinant Mbb.ruminantium A₁A_(o) ATP synthase was further tested in DK8 and nativemembranes. After preincubation with the inhibitor tributylin (TBT) for20 min at room temperature, the reaction was started at 39° C. byaddition of Na₂-ATP to a final concentration of 2.5 mM. 0.5 mg invertedmembrane vesicles was used in each end-point assay. Background ATPaseactivity generated by thermal hydrolysis of ATP or contaminant ATP inbuffer or enzyme was subtracted (these totalled <5% of the final valueshown; FIG. 31B).

ATP synthesis in E. coli DK8 inverted membrane vesicles was furtherevaluated. Time-course of ATP synthesis was assessed at pH 6.5, 125 mMNa⁺ and 39° C. with 0.5 mg of inverted membrane vesicles using the ATPsynthesis inverted membrane vesicle assay using NADH as a driving force.Membranes were preincubated for 2 min with 2.5 mM NADH with stirringbefore the reaction was initiated using 0.75 mM ADP and 2.5 mM Pi.Closed squares with no DCCD; closed triangles, a 20 min preincubationwith 250 μM TBT (FIG. 32).

Overview

We have successfully cloned, expressed and characterized the A₁A_(o)-ATPsynthase from Mbb. ruminantium. SDS-PAGE revealed 9 subunits and anSDS-stable K ring, which was purified. The A₁A_(o) synthase is active inboth synthesis and hydrolysis, but the enzyme activities can beimproved. This is consistent with other published A₁A_(o)-ATP synthasesfrom methanogens. The coupling ion for the enzyme is being identifiedwith studies suggesting both H⁺ and Na⁺ ions being important. ATPhydrolysis activity is sensitive to TBT, DCCD, and amiloride in highconcentrations, suggesting these inhibitors will be ineffective againstthe growth of methanogens, hence the need to find a better inhibitor.

We will proceed to test antibodies generated towards the A₁A_(o)-ATPsynthase in sheep against the purified enzyme in a Western blot. Theantibodies have been directed against the soluble A₁ part of the enzymeand therefore are probably inaccessible during growth inhibitionstudies. This suggests that targeting the A₁ portion will beineffective. We will also use the membrane-embedded sector (K ring) ofthe enzyme for new sheep antibody trials. This component would beaccessible in the whole cell, so could be very useful as a target. Wewill also use the recombinant enzyme to identify inhibitors of activityusing LOPAC1280™ which is a versatile compound library for assayvalidation and high throughput screening.

Example 5: Cloning and Expression of Non-Ribosomal-Peptide-Synthase(NRPS) Genes

Experiments are being performed to obtain expression of full-length NRPSgenes, isolate the expression product and submit for structuraldetermination and activity testing. Two non-ribosomal peptide synthetasegene sequences have been identified in M. ruminantium M1 (Leahy et al.,2010). We have been able to clone and amplify most of the functionalmodules of the NRPS1 gene of M. ruminantium. This allows us toinvestigate the substrate specificity and mode-of-action of individualNRPS domains. We have also completed the design and in vitro assessmentof predicted synthetic peptides from both the NRPS1 and NRPS2 geneproducts from M. ruminantium M1. The unexpected outcomes of theseexperiments (an opposed reaction to known siderophores) has promptedsignificant interest in the nature of these NRPs and their nativemolecular structure.

We have shown the presence of the native NRPs in M. ruminantium M1growth supernatant, and are obtaining further information on secondaryand tertiary native structures and on any further modification to thepeptide backbone, such as cyclisation or acylation. Therefore,expression of full length NRPS genes in a heterologous expression systemwill give us an opportunity to purify the active peptide compoundssynthesized and make the genes available for large scale production.

Suitable primer sets were designed to ensure full length amplificationand subsequent cloning into suitable expression systems. We haveobtained full length amplification of the M1 NRPS2 gene and are carryingout further experiments to clone this amplicon into a vector system.Small amounts of NRPS1 from M1 have been amplified and inserted intoentry vectors. These vectors are now being sequenced to confirm that theinserted amplicons reflect the NRPS gene and are free of non-silentnucleotide mutations.

We are also synthesizing NRPS genes using GeneArt's gene optimizationservice. This optimization not only adapts codon usage to theheterologous expression host E. coli but also accounts for factors thatmay compromise the stability of mRNA, such as extreme GC content,ribosomal binding sites, repeats and secondary structures. Substratefeeding studies with E. coli crude extract and E. coli//M. ruminantiumcrude extract mix will be carried out to evaluate the amount offunctional NRPS units. Biological active NRP molecules will subsequentlybe detected using the CAS colorimetric assay.

We are also working to induce gene expression by providing a range ofstimuli (i.e., increasing amounts of ion chelators) in the growth mediumand monitor gene expression levels. An alternative approach to isolatethe non-ribosomal peptides is to identify induction conditions of theNRPS genes via Northern Blot analyses. Induction of those genes willlead to increased mRNA levels, more active NRPS units and, subsequently,more NRP molecules in the growth supernatant. Based on our preliminaryresults reported earlier, we have identified two initial stressconditions to test. The interaction of NRP1 and 2 with Chrome Azure S(CAS) and Fe-ions, make iron scavengers and chelators such as Desferaland EDTA prime candidates for initial induction testing. The addition ofboth compounds to the growth medium may trigger the NRPS sensor systemand cause elevated gene expression.

For these experiments, M. ruminantium M1 was grown to an OD₆₀₀ of 0.1and then aliquoted in 5 mL amounts into anaerobic tubes. Differentconcentrations of EDTA and Desferal were added to the cultures. Eachculture was sampled at 1, 2, and 4 hours and overnight after theaddition of EDTA or Desferal. Samples were centrifuged to pellet thecells, supernatant was removed and the samples stored at −80° C.Subsequently, total RNA has been isolated and tested for integrity. Totest semi-quantitatively the level of gene expression, RNA dilutionseries for each sampled time point and each inducer concentration willbe established in a Northern Dot Blot system.

Three different probes have been designed targeting one house-keepinggene as positive control, and both NRPS genes. We are currently in theprocess of labelling these probes and will commence testing. Whenappropriate induction conditions have been established, we will purifythe native non-ribosomal peptides from M. ruminantium culturesupernatant using HPLC and assess for functionality using CAS assays.Furthermore, purified NRPs will be subjected to structural analyses werepossible. A comparison between the non-ribosomal peptides from M.ruminantium and those purified from the heterologous gene expressionsystem will allow upscaling the production of NRPs and derivativesthereof.

Example 6: Vaccination of Sheep Using Candidate Proteins Identified fromthe M. ruminantium Genome and Other Rumen Methanogens

Experiments are being performed to use up to ten selected gene targetsfrom M. ruminantium for heterologous expression, vaccinate sheep andtest resulting serum antibodies against methanogen cultures. The firststep in identifying candidate proteins for vaccine development is todetermine which M. ruminantium proteins are cell surface-located andpotentially accessible to antibody binding. In silico analysis of the M.ruminantium M1 open reading frames (ORFs) identified an initial pool of572 ORFs containing one or more transmembrane helices (TMH) or signalpeptide (SP) indicating a cell membrane or cell surface location. ThoseORFs with a top BLAST hit to a non-methanogen or with no homology to thenon-redundant database were removed and adhesin-like ORFs were dealtwith separately. This gave a new total of 337 ORFs. Examination of theremaining 337 ORFs, assessing their predicted function, degree ofconservation among methanogens and the nature of their transmembranestructures, refined the list to 71 ORFs (Leahy et al., 2010).Heterologous expression of membrane proteins with more than 4 TMHs hasbeen difficult in reverse vaccinology studies of other microbes, so acut-off of 4 THMs was applied to define two final groups: Group A with47 ORFs with 4 or fewer TMHs suitable for cloning and heterologousexpression studies; and Group B composed of 24 ORFs with more than 4TMHs more suitable for a synthetic peptide-directed vaccine approach(see below).

M. ruminantium Surface and Membrane Proteins Selected as Vaccine Targets

Functional Category and Locus tag Annotation Energy Metabolism mru0697AhaK mru1405*, 1406*, 1407, 1408, 1411, 1412 EhaHGFEAB mru2006, 2007,2008, 2010, 2012, 2013 EhblHGECB mru1917, 1918, 1921*, 1922*, 1923*MtrGFCDE Protein Fate mru0239 SecG mru0482 SecE mru1234* type IV leaderpeptidase family protein Vitamins & Cofactors mru0540 CbiN1 Hypotheticalmru0542*, 0840*, 1693, 2156*, 0233, 0234*, Hypothetical proteins 0330,1021, 1144, 1231, 1480*, 1585, 1635*, 1955, 2015*, 2046*, 2056, 2146*0529 0081, 0196, 0225*, 0328*, 0412, 0428*, 0499, 0596, 0597, 0693,0832, 1098, 1385, 0147, 0377*, 1375*, 1641, 0543, 0833, 1991, 0545*,0716*, 0718*, 0838, 0968, 1232, 1550, 1884*, 2202, 1694 *ORFs ≥ 5 TMHs

Many of these candidate genes correspond to proteins whose function isunder investigation. However, some are involved in energy production andare therefore prime candidates for vaccine development (FIG. 11). Ofparticular interest are the membrane-embedded ATP synthase enzymecomplex which generates ATP from either a sodium or proton gradient(Aha), the H₄MPT methyl transferase enzyme complex that catalyses thesecond to last step in the methanogenesis pathway (Mtr) and twomembrane-bound energy converting [Ni-Fe] hydrogenases (Eha and Ehb).

The H₄MPT methyl transferase enzyme complex is the penultimate step inthe methanogenesis pathway. The methanogenic archaea use this pathway togenerate energy (FIG. 11). The first five steps result in the sequentialreduction of CO₂ by electrons sourced from H₂ to form N5-methyl-H4MPT,then the methyl group is transferred to coenzyme M via the action of themethyl-H4MPT:CoM-methyltransferase. Mtr is made up of multiple enzymesubunits (Mtr E, D, C, B, A, F, G, H; FIG. 33) and couples the methyltransfer reaction to the efflux of Na⁺ ions out of the methanogen cell.This creates a Na⁺ gradient that is used, either directly, or via aNa⁺/H⁺ antiporter, to drive ATP synthesis. Our analyses indicate thatMtr subunits are sufficiently conserved and specific to methanogens tobe prime candidates for vaccine development. Therefore we aresub-cloning and expressing these subunits in order to obtain protein tovaccinate into sheep.

In order to clone the genes into the pTrc99A vector, 2 pairs of primerswere designed for each of the 9 target ORFs (8 individual subunits andcomplete operon-mtrEDCBEFGH). One set of primers introduces a His(6×Histidine) tag to the N-terminal of the translated proteins, whilethe other set of primers do not include this tag. The primers weredesigned for insertion of the fragments between the NcoI site and XbaIsite of the pTrc99A vector. Each open reading frame (ORF) from the mtroperon was amplified from M. ruminantium M1 genomic DNA, and ligatedinto the appropriately digested vector. The ligation products weretransformed into DH5=competent cells and insert-containing colonies wereselected and analysed to verify insertion of the correct gene. In orderto improve the chances of mtr gene expression, a codon-optimised mtrconstruct was designed for synthesis by GENEART (Germany) and clonedinto pTrc99A. Each mtr gene contains an RBS site, an N-terminal 6×Histag, a TEV protease cleavage site, and is flanked by unique restrictionsite compatible with sub-cloning individual synthetic ORFs into thepTrc99A.

The table below summarises the mtr gene cloning and expression results.All 9 ORFs were successfully amplified with the two pairs of primers,giving 18 constructs, of which 17 were successfully cloned into thepTrc99A vector. All constructs were sequenced to confirm correct geneinserts.

Mtr Cloning and Expression

Constructs Cloned Sequenced Expression mtrA-His  

  Yes  

  Yes In progress mtrB-His  

  Yes  

  Yes In progress mtrC-His  

  Yes  

  Yes  

  Yes mtrD-His  

  Yes  

  Yes In progress mtrE-His  

  Yes  

  Yes In progress mtrF-His  

  Yes  

  Yes In progress mtrG-His  

  Yes  

  Yes In progress mtrH-His  

  Yes  

  Yes  

  Yes mtrEDCBAFGH-His  

  Yes  

  Yes In progress mtrA  

  Yes  

  Yes In progress mtrB  

  Yes  

  Yes In progress mtrC  

  Yes  

  Yes In progress mtrD  

  Yes  

  Yes In progress mtrE  

  Yes  

  Yes In progress mtrF  

  Yes  

  Yes In progress mtrG  

  Yes  

  Yes In progress mtrH  

  Yes  

  Yes  

  Ye

Only the complete mtr operon without the 6×His tag failed to clone fromthe PCR product. Expression of all 17 mtr constructs has been obtainedusing the expression host OverExpress C41 (DE3) (Lucigen) cells grown inYT medium and induced by IPTG at either 37° C. or 30° C. MtrH with orwithout the 6×His tag was successfully over-expressed, and there alsoappeared to be some mtrH expression from the mtrEDCBAFGH-His construct(FIG. 34).

The MtrC construct with 6×His tag had low level expression in theOverExpress C43 (DE3) (Lucigen) expression host (FIG. 35). After lysisof the cells expressing the mtrH and mtrC proteins, the two proteinswere solubilised and put through the nickel columns for purification,but the proteins did not bind well to the columns, preventing columnpurification. The codon-optimised mtr synthetic construct has also beentested for expression in OverExpress C43 (DE3) (Lucigen) cells in YTmedium, and Rosetta II (DE3), OverExpress C43 (DE3), OverExpress C41(DE3), OverExpress C43 (DE3) pLysS, OverExpress C41 (DE3) pLysS cells inauto-induction medium (ZYP5052). No expression from any of the mtr geneswas detected under any of these conditions. Further expression ofcodon-optimised mtr synthetic construct in Rosettall (DE3) and inductiontemperature at 20 to 37° C. for a range of induction times (4 to 16hrs), with IPTG concentration ranging from 0.1 mM to 1 mM is beingassessed. We also plan to sub-clone each of the individual mtr subunitsfrom the synthetic construct and obtain expression individually.

Example 7: Vaccination of Sheep Using Cell Surface Protein FractionsIsolated from M. ruminantium Cells

Traditional vaccine development against bacteria relies on using cellfractions to elicit antigenic responses in the host animal. Thisapproach, while generally accepted, carries the inherent risk ofcontamination from other cell fractions. As an alternative totraditional cell fractionation, cell surface proteins isolated vianon-destructive treatment are investigated for their ability to createeffective and specific antigens. Chaotropic salts, such as guanidinehydrochloride, have been widely used to remove non-covalently boundproteins such as S-layers from the cell surface, while maintainingviable microbial cells.

In our experiments, M. ruminantium M1 cells have been subjected tochaotropic salt treatment removing non-covalently bound proteins fromthe cell surface. In a second step the treated methanogen cells havebeen subjected to a trypsin digest. This cleaved off exposed proteinepitopes from the cell surface, without disrupting the cell wall or cellmembrane. For this protocol, M. ruminantium M1 cells were grown in RM02media for 7 days. The culture was then centrifuged to spin down thecells and the supernatant discarded. The cell pellet was washed threetimes in sterile distilled water, resuspended in 2 mL of 4Mguanidine-HCl pH 7.0 and incubated at 37° C. for 1 hour. The suspensionwas centrifuged in a microfuge at 13 000 rpm. The supernatant(containing non-covalently bound surface proteins) was carefully removedand dialysed twice against 100 mM ammonium bicarbarbonate buffer (pH 8).A sample was run on an SDS-PAGE to estimate number and quantity ofisolated proteins.

These samples have been used in a sheep vaccination trial. A Westernblot analysis of M1 cell fractions against sheep antisera from the trialhas been carried out and a significant level of cross-reaction has beenobserved (FIG. 36).

A trypsin digest was then carried out for both the non-covalently boundisolated proteins and the exposed cell-surface epitopes. This digestalso served to prepare the samples for subsequent MALDI-TOF analyses.Trypsin was added at an approximate ratio of 1:50 relative to estimatedamount of protein (based on SDS-PAGE). 10% (v/v) HPLC grade acetonitrilewas added and the samples were incubated at 37° for 12 hours. Thedigests were centrifuged, supernatants were collected and subsequentlydialysed against 100 mM ammonium bicarbonate buffer (pH 8). Whereappropriate, DTT (dithiothreitol) was added to a final concentration of50 mM and the samples were incubated at 60° C. for 30 minutes to reducedisulphides. IAM (iodoacetamide) was then added to a final concentrationof 150 mM and samples were incubated in the dark at room temperature for30 minutes. DTT and IAM were removed from the sample by dialysis orwashing with 100 mM ammonium bicarbonate buffer (pH 8). Bothpreparations were sent to Dr Stefan Clerens (AgResearch, Lincoln) forMALDI-TOF. Protein fragments were subsequently compared to thenon-redundant amino-acid database hosted by NCBI and a custom ORFeomebased M. ruminantium database (Leahy et al., 2010), associating epitopeswith their respective ORFs.

Six independent culture trials were conducted, including cells fromnormal growth conditions, cells from medium supplemented with methanoland cell subjected to oxygen stress. A total of 57 fragments could beassigned to their respective M. ruminantium M1 genes (see table, below).It is noteworthy that a significant level of variety in identified ORFswas encountered in between the individual protein isolations. This maybe caused by a sensitive and rapid adaptation of M. ruminantium to evensmall changes in culture conditions. Notable targets such asadhesion-like proteins or an ABC transporter substrate-binding proteinwere detected which will be merged into the priority target list.Interestingly, a number of cytosolic proteins (i.e., ribosomal proteinsand other predicted highly expressed genes) were also detected,indicating a certain amount of ongoing cell lysis during the samplepreparation. In this context, we also identified the M1 bacteriophageφmru integrase protein, pointing to a continued phage lysis process.This is supported by the detection of φmru particle in culturesupernatant during normal growth conditions. The active prophage notonly points to a highly stressed M. ruminantium cell condition but alsomay cause an altered biochemical and phenotypical profile.

It therefore is important to improve culture conditions and also developa prophage-free strain that lacks the potential of accelerated anduncontrolled cell lysis. Consequently, we have initiated a phage curingexperiment which aims to create a phage-free M. ruminantium derivative.Because M. ruminantium cannot be cultivated on solid media traditionalphage curing methods cannot be employed. We have therefore opted for anevolutionary approach that uses ProteinaseK to remove free phageparticles and continuous minimal subculturing. We will enrich for aprophage-free M. ruminantium population that eventually evolves in afully cured derivative. This derivative would then also be used infurther experiments, such as the assessment of the prophage φmru. Mostinterestingly, we were also able to identify the M1 NRPS1 gene product,indicating that the NRPS system can be active and expressed underlaboratory growth conditions.

List of Open Reading Frames Identified by MALDI-TOF Analyses.

ORF number Annotation Count 82 Adhesin-like protein+ 1  113 (1228)Exopolysaccharide biosynthesis polyprenyl 1glycosylphosphotransferase{circumflex over ( )} 115 RadA DNA repair andrecombination protein+ 1 117 HdrA CoB-CoM heterodisulfide reductasesubunit A*** 7 201 ModA molybdate ABC transporter substrate-bindingprotein+ 1 205 Copper ion binding protein{circumflex over ( )} 1 247ThiC1 thiamine biosynthesis protein*** 7 256 Phage integrase*** 5 326Adhesin-like protein*** 2 333 FdhA1*** 3 334 FdhB1*** 3 351 Nrps1+ 1 455Acetyltransferase+ 1 481 FtsZ cell division protein+ 1 498 Fbp fructose1,6-bisphosphatase*** 3 520 Translation elongation factor aEF-1 beta+ 1526 Hmd coenzyme F420-dependent N(5),N(10)- 5methenyltetrahydromethanopterin reductase*** 550 PorA pyruvateferredoxin oxidoreductase alpha subunit*** 4 551 PorB pyruvateferredoxin oxidoreductase beta subunit*** 2 569 Mer5,10-methylenetetrahydromethanopterin reductase*** 7 582 PhoU phosphateuptake regulator PhoU*** 2 595 PurP+ 1 629 Hypothetical protein+ 1 735Rbr1+ 1  727 (2191) Adhesin-like protein with cysteine proteasedomain{circumflex over ( )} 1 774 Archaeal histone+ 1 816 HdrC CoB-CoMheterodisulfide reductase subunit C*** 6 817 HdrB*** 3 851 Rpl3pribosomal protein L3P+ 1 872 Rps5p ribosomal protein S5P+ 1 949 BtcCbicarbonate ABC transporter substrate-binding protein*** 8 1052Transcriptional regulator+ 1 1091 SucD{circumflex over ( )} 2 1490Ribosomal protein L7Ae+ 1 1228 (113) Hypothetical protein{circumflexover ( )} 1 1371 Hypothetical protein+ 1 1491 Archaeal histone*** 8 1499Adhesin-like protein with transglutaminase domain+ 1 1570 Acs,ADP-dependent acetyl-CoA synthetase*** 5 1645 Thermosome subunit*** 21683 Hypothetical protein*** 3 1686 Archaeal histone*** 2 1691 MoaAmolybdenum cofactor biosynthesis protein+ 1 1730 Heat shock proteinHsp20+ 1 1805 Translation elongation factor aEF-1 alpha*** 7 1836 Cellshape determining protein MreB/Mr1 family*** 3 1888 PycB+ 1 1897 PpsA1phosphoenolpyruvate synthase+ 1 1900 Hypothetical protein+ 1 1901Peptidyl-prolyl cis-trans isomerase+ 1 1906 MvhA methylviologen-reducing hydrogenase alpha*** 7 1907 MvhG methylviologen-reducing hydrogenase gamma*** 4 1916 MtrHtetrahydromethanopterin S-methyltransferase*** 9 1919 MtrA1tetrahydromethanopterin S-methyltransferase*** 4 1920 MtrBtetrahydromethanopterin S-methyltransferase+ 1 1921 MtrCtetrahydromethanopterin S-methyltransferase*** 2 1924 McrAmethyl-coenzyme M reductase alpha subunit*** 5 1925 McrG methyl-coenzymeM reductase gamma subunit*** 12 1928 McrB methyl-coenzyme M reductasebeta subunit*** 9 1993 CBS domain-containing protein+ 1 1994 CBSdomain-containing protein*** 8 2022 Ftr2formylmethanofuran-tetrahydromethanopterin 7 formyltransferase*** 2074FdhA2 formate dehydrogenase alpha chain+ 1 2110 IlvC ketol-acidreductoisomerase+ 1 2121 Hcp hydroxylamine reductase*** 5 2131 Fae/Hpsbifunctional formaldehyde-activating enzyme/3-hexulose-6- 1 phosphatesynthase+ 2142 Mtd F420-dependent methylenetetrahydromethanopterin 11dehydrogenase*** 2159 TrpB2 tryptophan synthase beta subunit*** 62191(727) CDP-glycerol:poly(glycerophosphate)glycerophosphotransferase{circumflex over ( )} 1 ORFnumber indicates therespective locus tag within the M. ruminantium M1 genome; Annotationshows the corresponding functional annotation; Count highlights thenumber of occurrences found for each ORF throughout the six individualruns. ***ORFs found multiple times, +unique hits, {circumflex over( )}Hits with sequence mismatch to M1 deduced aa sequences. ORF numbersin brackets indicate ambiguous hits.

Example 8: Expression and Purification of Enzymes from the Methanogen M.ruminantium for the Discovery of Inhibitors

Novel inhibitors have great potential to provide mitigation of thegreenhouse gas methane from ruminants. This area of research hassignificance in the stabilisation of greenhouse gas concentrations inthe atmosphere to prevent climate change. The principal methane formingorganisms in the rumen are archaea belonging to the genusMethanobrevibacter. We are targeting genes of rumen methanogens forcloning and expression in E. coli with the aim of obtaining purifiedenzymes that can be used for: high-throughput screening (HTS) assays ofchemical compound libraries; and in silico screening of inhibitors. Themajority of the targets are from five main functional classes;methanogenesis/energy metabolism, central carbon metabolism, cofactorsynthesis, cell wall synthesis and lipid synthesis. Work will continueto advance the targets through the pipeline. This project bringstogether diverse disciplines including chemogenomics, in silicomodelling, structural biology, and the in vitro biological screening oftargeted compounds to formulate the development of potentanti-methanogen compounds that are non-toxic to host ruminant animalsand have negligible environmental impact.

Our aim is to discover small molecule inhibitors of methanogens, basedon genomics, biochemistry, and structural biology. This is a powerfulmeans to search for inhibitors of methanogens, which are difficult toculture and are not amenable to high-throughput screening with cells. Inthis study, a number of enzymes from Methanobrevibacter ruminantium werefound to be solubly expressed in E. coli e.g.3-hydroxy-3-methylglutaryl-CoA reductase (HMGR),methenyltetrahydromethanopterin cyclohydrolase (MCH),3-hexulose-6-phosphate isomerise (PHI) and bifunctionalformaldehyde-activating enzyme/3-hexulose-6-phosphate synthase(FAE/HPS). HMGR catalyzes the rate-limiting step in the synthesis ofisoprenoid units, which are components of archaeal membrane lipids. MCHis an enzyme involved in the methanogenesis pathway. PHI and FAE/HPS arekey enzymes of the ribulose monophosphate pathway, used by methanogensto generate ribose for nucleotide synthesis.

As a first step, we made a selection of targets. Information fromliterature and genome of Methanobrevibacter ruminantium (Leahy et. al.,2010) was studied and 34 targets (see table in Example 9, below) werechosen. Next, we carried out cloning in E. coli. Genes were amplifiedand cloned into expression vector pET151D (Invitrogen). Plasmids wereused to transform E. coli BL21-Rosetta 2 cells (Novagen). From this, 27positive clones (see table, below) were obtained. We then looked forexpression of recombinant proteins. Cells were grown in auto inductionmedium ZYP-5052 (Studier, 2005), with shaking at 25° C. or 30° C. forapprox 16 hr. Cells were lysed using lysozyme at 4° C. Thehexa-histidine-tagged enzymes were then purified from cell free extractsby nickel-affinity chromatography. Imidazole was removed and buffer wasexchanged. We found 15 proteins were expressed while 10 (12) weresoluble (see table, below). Clones that failed to express were grown inLB media induced with IPTG. Clones with insoluble expression weresubjected to varying lysis conditions. To overcome the lack ofexpression in some clones, the genes have been synthesised for optimumexpression in E. coli (GeneArt).

Biochemical characterisation of HMGR has also been carried out. Thisincluded assays to measure the oxidation of NADHP (366 nm, E 3,300 M-1cm-1), which were performed at 37° C. Activity was expressed in U mg-1of enzyme. One unit was defined as the turnover of one μmol of NADPH perminute (2 NADPH molecules are required to reduce 1 HMG-CoA). Standardassays contained 50 mM BTP (Bis-Tris propane) pH 6.5, 400 mM NaCl, 0.05mM DTT, 2.5% glycerol, 338 μM NADPH and 250 μM (R,S)-HMG-CoA. Prior touse, the enzyme stock (0.6 mg/mL) was incubated in 400 mM NaCl and 10 mMDTT for 2 hours at 4° C. and for 20 minutes at 37° C. then kept on ice.Assays were carried out in duplicate. HMGR was susceptible to oxidationand could be reactivated by incubation with 10 mM dithiothreitol for twohours. Highest activity was found at pH 6.5 and at 0.4-1.5 M NaCl. HMGRwas able to oxidize NADPH but not NADH. The enzyme had Km values of165±35 and 12.4±1.83 μM for NADPH and HMG-CoA, respectively. The statinssimvastatin and lovastatin inhibit HMGR with high Kic values of 3.7±0.65and 8.5±1.1 μM, respectively. Statins have previously been shown toinhibit the growth of strains of rumen Methanobrevibacter (Wolin andMiller, 2006). Notably, structure-based alignment showed that the enzymeis a Class I HMGR.

Example 9: In Silico Modelling of Enzymes from the MethanogenMethanobrevibacter ruminantium for the Discovery of Novel Inhibitors

Analysis of the Methanobrevibacter ruminantium genome (Leahy et. al.,2010) has revealed archaeal and methanogen-specific enzyme pathwaysinvolved in methane production, energy metabolism, protein, lipid,cofactor and cell wall synthesis. Making comprehensive use of thisgenomic data, essential protein targets have been analysed based onalready available structural data of related enzymes for homologymodelling, or when possible, purified protein has been submitted toprotein crystallisation trials for x-ray structure determination.

The presence of published crystal structures that are similar insequence is a factor for selecting targets, due to the fact that theycan be used to guide the determination of our own crystal structures,thus saving time. In addition, high-resolution structures can also beused as models to develop inhibitors, as is now being performed. Forexample, ODcase (orotidine-5′-phosphate decarboxylase; PyrF) is a keyenzyme in the biosynthesis of the pyrimidine uridine-5′-monophosphate(UMP) (Nyce and White 1996). ODcase from other sources have providedcrystal structures and high-quality work on developing inhibitors aspart of its kinetic characterisation (Wu and Pai, 2002; Poduch et al.,2006; Poduch et al., 2008; Fujihashi et al., 2009; Bello et al., 2007).Other researchers are using ODcase for the development of novelanti-pathogenic compounds based on small differences between thepathogen and mammalian structures (Bello et al., 2007). There are over40 crystal structures for methanogen ODcases. Similarly, HMG CoAreductase from other sources has been identified as the presumed targetof statins (Miller and Wolin, 2001). The work of Miller and Wolinvalidated HMG CoA reductase as a target in methanogens, and indeed, theentire mevalonate pathway for lipid synthesis. There are >20 HMG CoAreductase crystal structures available.

The target sn-glycerol-1-phosphate dehydrogenase (NAD(P)-dependentglycerol-1-phosphate dehydrogenase, EgsA) is a well known archaealenzyme that forms the stereo-specific glycerol-1-phosphate backbone forarchaeal lipids (Koga and Morii, 2007). GGPS (geranylgeranylphosphatesynthase) is another lipid synthesis enzyme that is being targeted as italso has an archaeal stereospecific catalytic site (Koga and Morii,2007). A crystal structure is available for a methanogen GGPS (Payandehet al. 2006). As another example, DAPDC (diaminopimelate decarboxylase;LysA) helps catalyse the formation of lysine which is a keycross-linking component of M. ruminantium cell walls. Lysine is anessential amino acid in mammals and therefore the lysine biosynthesispathway has been targeted for the development of novel antibiotics(Hutton et al., 2007). A crystal structure is available for DAPDC (Rayet al., 2002). The methanopterin biosynthesis pathway enzyme RFA-Psynthase has been validated as a target in other systems, and a vastarray of potential inhibitors have been identified (Dumitru et al.,2003; Dumitru and Ragsdale, 2004; Scott and Rasche, 2002; Miner et al.,2003). The methanopterin pathway enzyme CitG is presumed to be essentialand is nearly-methanogen specific (Chistoserdova et al., 2003,Chistoserdova et al., 2004, Schneider et al., 2000). HisAF is alsoindicated to be part of the methanopterin pathway (Chistoserdova et al.,2003, 2004).

The F420 biosynthesis pathway includes the targets of CofA, PLT (CofD),CofC (PLGT), and creatinine amidohydrolase (CA) and FucA are nearlymethanogen-specific and presumed to be essential for methanogen survival(Graham and White 2002). Most of the remainder of the F420 pathway isalso being targeted. A crystal structure is available for PLT (Forouharet al. 2008). Several methanogenesis pathway enzymes are included whichare encoded by single genes. These are essential for survival and thereare crystal structures for all four (Hiromoto et al., 2009; Shima etal., 2008; Grabarse et al., 1999; Aufhammer et al., 2005; Hegemeier etal., 2003). The Coenzyme M (CoM) pathway is also thought to be essentialfor survival and crystal structures are available for ComA and ComC(Irimia et al. 2004; Wise et al. 2003). The Coenzyme B biosynthesispathway is the only methanogen cofactor pathway that is fullymethanogen-specific and should be absolutely essential (Graham andWhite, 2002). Despite this, all the known enzymes (AksA, AksD, AksE andAksF) share significant homology with bacterial proteins, and three havesome homology with mammalian enzymes (AksD, AksE and AksF have somehomology with mammalian aconitase subunits and/or isocitratedehydrogenase).

The RuMP, or ribulose monophosphate pathway, is used by methanogens togenerate ribose for nucleotide synthesis. Although it is notmethanogen-specific it is only found in a very limited number oforganisms and these are not typically found in the rumen (Werken et al.,2008; Growchowski and White, 2005). Several of the key enzymes of thepathway are of interest, and in M. ruminantium two of these are linkedto form a bifunctional enzyme (HPS-FAE, formaldehyde-activatingenzyme/hexulose-6-phosphate synthase). Phil (hexulose-6-phosphateisomerase) is also being targeted and is also part of the RuMP pathway.Interestingly, M. ruminantium also has another Phi (Phi2), althoughsequence analyses suggests that Phil is the more likely to be involvedin the RuMP pathway. A methanogen crystal structure is available for Phi(Martinez-Cruz et al., 2002).

The targets Mur 53, 78, 520, 873 and 874 are murein ligases involved insynthesising the amino acid peptide linkages of the cell walls ofmembers of the Methanobacteriales. Murein ligases are also found inbacteria. Disruption of cross-linking peptide biosynthesis would be akinto discovering an ‘anti-methanogen antibiotic’ with similar effects topenicillin-like drugs (Zoeiby et al., 2003; Hartmann and König, 1990).Most of these methanogen murein ligases (53, 520, 873 and 874) are quitedistinct from their bacterial homologues, whereas Mur 78 retains quitehigh levels of homology with its bacterial counterparts. Interestingly,Mur 520 is also found in some Methanococci and Methanosarcina. Bacterialmurein ligase structures (>40) are available that could be used asscaffolds for performing molecular replacement, thus aiding futurestructure determination of the methanogen enzymes.

Several enzymes are targeted as they are considered ‘key’ enzymes thatare central to metabolism (gluconeogenesis that is required for aminoacid, cell wall sugar synthesis, DNA and RNA synthesis and the TCAcycle), and therefore essential. Most of these have relatively easyspectrophotometric assays (Acs, PEP syn, AcsA, SdhA/SdhB). Acs andSdhA/B have archaeal-specific features (Bobik and Wolfe, 1989; Musfeldtand Schonheit, 2002). GatD and GatE are involved in translation and arearchaeal-specific and provide glutaminyl-tRNA for protein synthesis(Possot et al. 1988). tRNA synthetases have been successfully targetedfor the development of novel antibiotics (Ahel et al. 2005). There is amethanogen crystal structure for GatD/E (Oshikane et al. 2006).

The current targets (see below) represent 12 different cellularprocesses or metabolic pathways with a large share (16 targets) beingderived from methanogen cofactor synthesis pathways. Taking everythinginto consideration, methanogen cofactors represent strong targetsoverall as they are typically restricted to methanogens, are likely tobe essential and the cofactors themselves are fairly small molecules.Advantages of small molecules are that it can be easier to synthesisesubstrates for enzyme assays and easier to synthesise potentialinhibitors that share similar structural features. Due to potentialoff-target effects, we have decided to avoid most vitamin synthesispathways as almost all of the methanogen enzymes have counterparts inbacteria and therefore, inhibitors would have a high chance of alsoinhibiting beneficial rumen bacteria.

Current Chemogenomics Targets

expressed/ Target pathway PCR clone soluble Pure X-tal comments ODcase(PyrF) pyrimidines no early target HMG CoA red lipids yes yes yes/yesyes yes early target sn-G1P deh2 lipids yes yes no GA; early targetDAPDC (LysA) lysine/CW yes yes yes/yes yes early target RFA-P (MptG)methanopterin yes yes yes/no GA PLT (CofD) F420 yes yes no GA CofA(1093)F420 yes yes GA FucA F420 yes yes yes/yes Phi1 RuMP yes yes yes/yes yesyes Hmd methanogenesis yes yes no GA Mch methanogenesis yes yes yes/yesyes yes Mer methanogenesis yes yes yes/no GA Mtd methanogenesis yes yesGA AksA Coenzyme B yes AksD Coenzyme B yes yes yes/no AksE Coenzyme Byes AksF Coenzyme B yes yes no CitG methanopterin yes yes yes/yes ComA(Homolog) coenzyme M yes yes yes/yes ComB (Homolog) coenzyme M yes yesno GA ComC (Homolog) coenzyme M yes yes yes/yes yes ComD (Homolog)coenzyme M yes yes no GA ComE (Homolog) coenzyme M yes yes no GA Hps-FaeRuMP yes yes yes/yes yes yes Acs key enzyme yes AcsA key enzyme yes PEPsyn key enzyme yes yes yes/yes GGPS lipids yes yes yes/some SdhA TCA yesSdhB TCA yes Mur 53 cell wall yes yes GA Mur 78 cell wall yes yesyes/some yes Mur 520 cell wall yes yes Mur 873 cell wall no GA Mur 874cell wall no GA GatD translation yes yes GatE translation yes CofC F420no CA F420/riboflavin no HisAF methanopterin no GA, gene synthesised byGeneArt and will be recloned; CA, creatinine amidohydrolase

For specific experiments, in silico screening of large commercialcompound libraries using the docking programme GOLD (Verdonk et. al.,2003; Cole, J. C. et. al., 2005) has formed the basis of our selectionand design of high affinity ligands for our modelled proteins. Work iscurrently underway for evaluating the essential methanogen enzymemethyl-coenzyme M reductase (MCR), an enzyme that catalyzes the terminalstep in methane production. A number of enzymes are being crystallisedand include 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), therate-limiting step in the synthesis of isoprenoid units, which arecomponents of archaeal membrane lipids. Others include3-hexulose-6-phosphate isomerase (PHI), a key enzyme of the ribulosemonophosphate pathway and methenyltetrahydromethanopterin cyclohydrolase(Mch), an enzyme involved in the methanogenesis pathway.

The programme GOLD (Verdonk et. al., 2003; Cole, J. C. et. al., 2005)has been tasked in carrying out the in silico docking process in orderto obtain novel inhibitors of archaeal and methanogen-specific enzymes,in particular MCR (Grabarse et. al., 2001; FIG. 37A). MCR has an α2β2γ2subunit structure which contains two nickel porphinoid F430 rings whichforms the centre of enzyme activity and two molecules each ofmethylcoenzyme M (CoM) and coenzyme B (CoB). These cofactors operateunder strictly anaerobic conditions to carry out the final reaction ofthe energy conserving pathway of methanogenic archaea in which CoM andCoB are converted to methane and the heterodisulfide product CoM-S-S-CoB(FIG. 37B). This product is subsequently reduced with H2 back to thethiol forms of the separate cofactors by heterodisulfide reductase.

A number of structural databases from commercial suppliers have beendownloaded from the ZINC (Irwin and Shoichet, 2005) website hypertexttransfer protocol://zinc.docking.org/index.shtml and screened with GOLD(Verdonk et. al., 2003; Cole, J. C. et. al., 2005). Commercial databasesavailable from the zinc website such as Asinex, Chembridge buildingblocks and the LOPAC library of proven pharmacologically-activecompounds were screened with MCR. Utilizing a ruminant model of MCRbased on a crystal structure from Methanothermobacter marburgensis (pdbcode 1HBM4) active site regions were subjected to specific and targeteddocking attempts to find inhibitors that could mimic the naturalsubstrates and product of the enzyme. The effectiveness of candidateinhibitors were then monitored in pure culture experiments. Cell densitywas measured over time to assess the effectiveness of potentialinhibitors (FIG. 38).

TABLE 1 Comparison of the M1 genome features with methanogens from theorder Methanobacteriales M. ruminantium M. smithii M. smithii M. smithiiMethanothermobacter Methanosphaera M1 PS [34] ALI^(a) F1^(a)thermoautotrophicus ΔH [46] stadtmanae MCB-3 [20] Source Bovine rumenSewage digester Human Human faeces Sewage sludge Human faeces Projectstatus complete complete draft draft complete complete Genome size (bp)2,937,203 1,853,160 1,704,865 1,707,624 1,751,377 1,767,403 G + Ccontent (%) 33 31 31 31 50 28 Number of ORFs 2217 1795 1709 1710 18731534 Coding area (%) 81 90 90 90 90 84 rRNA operons 2 2 nd nd 2 4 tRNAs(with intron) 58 (2) 34 (1) 34 34 39 (3) 40 (1) Non-coding RNA 3 3 nd nd2 2 Insertion 4 8 nd nd 0 4 sequences Prophage Yes Yes nd nd No NoCRISPR regions 2 1 nd nd 2 2 Adhesin-like 105 48 nd nd 0 37 proteinsLPxTG motif 1 2 nd nd 0 0 Sortases 1 1 nd nd 2 0 ^(a)Draft genome dataobtained from National Centre for Biotechnology Information hypertexttransfer ptotocol://www.ncbi.nlm.nih.gov/; nd Data not determined fromdraft genome [34] Boekhorst et al., 2005 [46] Smith et al. 1997 [20]Fricke et al., 2006

TABLE 2 Potential chemogenomic gene targets of the M1 genome based onin-depth literature and metabolic analyses. Locus Annotation ReferenceAMINO ACID METABOLISM mru0997 phospho-2-dehydro-3-deoxyheptonatealdolase/ [S8, 9] fructose-bisphosphate aldolase mru0998 AroB [S9-11]mru1577 AroA [S9, 10, 12] mru1676 AroK [S13] mru0350 GlnA1 [S14] mru2078GlnA2 mru0122 GlyA [S15-17] mru2139 HisB [S18] mru0152 LysA [S19-21]mru0153 DapF [S21, 22] mru1743 PdaD [S23, 24] mru0208 TrpE [S25] mru0410IIvB1 [S26, 27] mru2112 IIvB2 mru2111 IIvN mru1414 CimA [S28, 29] CELLCYCLE mru0481 FtsZ [S30, 31] mru0240 PolD2 [S32] mru2212 PolD1 mru1864DNA topoisomerase VI subunit A [S33, 34] mru1865 DNA topoisomerase VIsubunit B CELL ENVELOPE mru0824 adhesin-like protein withtransglutaminase [S35-44] mru0828 domain mru1497 adhesin-like proteinwith transglutaminase mru1499 domain mru1604 adhesin-like protein withtransglutaminase domain adhesin-like protein with transglutaminasedomain adhesin-like protein with transglutaminase domain mru1836 cellshape determining protein MreB/Mrl family [S45-47] mru1047poly-gamma-glutamate biosynthesis protein [S48, 49] mru2175 cell wallbiosynthesis glycosyl transferase mru0707 cell wall biosynthesis proteinMur ligase family [S50-54] mru1042 cell wall biosynthesis protein Murligase family mru1118 cell wall biosynthesis protein Mur ligase familymru1745 cell wall biosynthesis protein Mur ligase family mru2091 cellwall biosynthesis protein Mur ligase family mru2092 cell wallbiosynthesis protein Mur ligase family mru0964 cell wall biosynthesisprotein phospho-N- [S55-57] mru1041acetylmuramoyl-pentapeptide-transferase family cell wall biosynthesisprotein phospho-N- acetylmuramoyl-pentapeptide-transferase familymru2126 cell wall biosynthesis protein UDP- glycosyltransferase familymru1293 GlmS1 [S52, 58] mru1536 GlmS2 mru1388 NAD dependentepimerase/dehydratase [S59] mru1413 NAD dependent epimerase/dehydratasemru0458 GlmM1 [S52, 58] mru0449 GlmM2 mru1733 phosphosugar-bindingprotein mru2136 polysaccharide biosynthesis protein mru1470 GalE [S59]mru0456 GlmU [S52, 58] mru1005 UppS [S60, 61] mru2108 UppP [S52, 62-64]mru1524 polysaccharide biosynthesis protein [S65] CENTRAL CARBONMETABOLISM mru1434 AcsA [S66] mru1570 Acs [S66-68] mru0550mru PorA[S69-74] 0551 PorB mru0549 PorD mru0548 PorD mru0552 PorE mru0553 PorFmru0957 RpiA [S75-77] mru1634 Prs [S78] mru0250 Phi1 [S75, 77, 79-mru1310 Phi2 81] mru2131 Fae/Hps [S75-77, 81, 82] mru1255 Mdh [S83-86]mru0847 PycA [S87-90] mru1888 PycB mru0088 SdhA [S91, 92] mru0655 SdhBENERGY METABOLISM mru0701 AhaA [S93-105] mru0702 AhaB mru0699 AhaCmru0703 AhaD mru0698 AhaE mru0700 AhaF mru0695 AhaH mru0696 AhaI mru0697AhaK mru2064 FrhA [S106, 107] mru2061 FrhB1 mru2081 FrhB2 mru2063 FrhDmru2062 FrhG mru1412 EhaA [S108, 109] mru1411 EhaB mru1410 EhaC mru1409EhaD mru1408 EhaE mru1407 EhaF mru1406 EhaG mru1405 EhaH mru1404 EhaImru1403 EhaJ mru1402 EhaK mru1401 EhaL mru1400 EhaM mru1399 EhaN mru1398EhaO mru1397 EhaP mru1396 EhaQ mru1394 EhaR mru2014 EhbA [S108, 110]mru2013 EhbB mru2012 EhbC mru2011 EhbD mru2010 EhbE mru2009 EhbF mru2008EhbG mru2007 EhbH mru2006 EhbI mru2005 EhbJ mru2004 EhbK mru2003 EhbLmru2002 EhbM mru2001 EhbN mru2000 EhbO mru1999 EhbP mru1998 EhbQ mru1906MvhA [S111-113] mru1905 MvhB mru1908 MvhD1 mru2076 MvhD2 mru1907 MvhGmru0569 Mer [S106, 114- 116] mru0117 HdrA [S106, 115, mru0817 HdrB117-122] mru1212 HdrB2 mru0816 HdrC mru0526 Hmd [S106, 123- 127] mru2142Mtd [S126, 128- 131] mru1393 Ftr1 [S106, 115- mru2022 Ftr2 116, 132-133]mru1619 Mch [S106, 114, 115, 129, 134-136] mru1924 McrA [S106, 114,mru1928 McrB 115, 137-151] mru1926 McrC mru1927 McrD mru1925 McrGmru1262 AtwA1 mru1850 AtwA2 mru1919 MtrA1 [S106, 115, mru0441 MtrA2 121,152-155] mru1920 MtrB mru1921 MtrC mru1922 MtrD mru1923 MtrE mru1918MtrF mru1917 MtrG mru1916 MtrH mru0344 FwdA S121, 156- mru0343 FwdB 162]mru0345 FwdC mru0342 FwdD mru0254 FwdE mru0340 FwdF mru0341 FwdG mru0339FwdH LIPID METABOLISM mru1031 FabG1 [S163-167] mru1630 FabG2 mru0955EgsA [S77, 168, 169] mru1092 HmgA [S170-175] mru1640hydroxymethylglutaryl-CoA synthase [S169, 172, 173, 176, 177] mru0922Fni [S77, 176, 178-180] mru0921 isopentenyl diphosphate kinase [S77,181] mru0920 Mvk [S170-175] mru0919 phosphomevalonate decarboxylase[S77] mru1102 digeranylgeranylglyceryl phosphate synthase [S182] mru0924IdsA [S177] MOBILE ELEMENTS mru0317 phage-related protein [S35] mru0320endoisopeptidase PeiR [S171] PROTEIN FATE mru2021 transglutaminasedomain-containing protein [S35] mru0391 oligosaccharyl transferasemru1832 sortase family protein [S183, 184] PROTEIN SYNTHESIS mru2169GatA [S185-189] mru2029 GatB mru1142 GatC mru1571 CysS [S185, 186, 190]mru1427 GatD [S185-187, mru1426 GatE 191-193] mru0126 IleS [S186, 194-196] mru0242 LysS [S185-187] mru0954 ProS [S197, 198] mru1947 SerS[S186, 199, 200] PURINES AND PYRIMIDINES mru1839 PurO [S77, 201, 202]mru0595 PurP [S77, 203] mru1055 PyrF [S204, 205] TRANSCRIPTION mru1482RpoE1 [S206, 207] mru1481 RpoE2 mru1815 RpoA1 mru1814 RpoA2 mru1816RpoB1 mru1817 RpoB2 mru0908 RpoD mru0161 RpoF mru1818 RpoH mru0913 RpoKmru0169 RpoL mru0912 RpoN mru1350 RpoP TRANSPORTERS mru0405 transporterNa+/H+ antiporter family [S208] UNKNOWN FUNCTION mru0668 methanogenesismarker protein 1 [S209] mru1929 methanogenesis marker protein 10 mru0097methanogenesis marker protein 11 mru0181 methanogenesis marker protein13 mru1915 methanogenesis marker protein 14 mru1771mru methanogenesismarker protein 15 1778 methanogenesis marker protein 2 mru1774methanogenesis marker protein 3 mru1931 methanogenesis marker protein 7mru0436 methanogenesis marker protein 8 mru1695 H4MPT-linked C1 transferpathway protein VITAMINS AND COFACTORS mru1560 HemB [S153, 210- 212]mru1541 CobA [S153, 210- 212] mru1853 HemA [S153, 210- 215] mru1544 HemD[S153, 210- 212, 216] mru0999 HemL [S210-214] mru1746 HemC [S153, 211-212] mru0384 AksD [S217-219] mru1689 AksE [S217-219] mru0385 AksA [S217,219, 220] mru1033 AksF [S217, 219, 221] mru1283 ArfB [S222] mru0953 CofC[S217, 223] mru1253 FtsA1 [S217, 224- mru1787 FtsA2 226] mru0479 F420-0:gamma-glutamyl ligase [S217] mru1842 CofE [S217, 227- 229] mru1974 CofG[S230-232] mru1266 CofH mru2213 FucA [S233-236] mru0672 CofA [S235]mru1844 CofD [S237, 238] mru1949 ComB [S239, 240] mru1980 ComC [S82,217, 241, 242] mru1896 MfnA [S217, 243] mru1690 MptG [S244-249] mru1962MptA [S250, 251] mru1559 CitG [S252-254] mru1845 ArfA [S255, 256]mru1215 RibC [S257-262] mru0098 RibH [S257-259] mru1007 RibD [S263]mru2174 RibK [S264, 265] S8 White & Xu, 2006 S9 Porat et al., 2006 S10White, 2004 S11 Porat et al., 2004 S12 Morar et al., 2007 S13 Daughertyet al., 1999 S14 Possot et al., 1998 S15 Lin & Sparling, 1998 S16 Hoytet al., 1986 S17 Angelaccio et al., 2003 S18 Sment & Konisky, 1989 S19Hutton et al., 2007 S20 Born & Blanchard, 1999 S21 Girodeau et al., 1986S22 Pillai et al., 2006 S23 Tolbert et al., 2003 S24 Graham et al., 2002S25 Kalyazhnaya et al., 2005 S26 Xing & Whitman, 1987 S27 Tan et al.,2006 S28 Hernández-Montes et al., 2008 S29 Howell et al., 1999 S30 Huanget al., 2007 S31 Löwe & Amos, 1998 S32 Ishino & Cann, 1998 S33 Grailleet al., 2008 S34 Gadelle et al., 2005 S35 Makarova et al., 1999 S36Esposito et al., 2007 S37 Griffin et al., 2002 S38 Yokoyama et al., 2004S39 Iranzo et al., 2002 S40 Kato et al., 2008 S41 Hartmann & Konig, 1990S42 Lee et al., 1985 S43 Luo et al., 2002 S44 Steenbakkers et al., 2006S45 Divakaruni et al., 2007 S46 Osborn & Rothfield, 2007 S47 Daniel &Errington, 2003 S48 Candela & Fouet, 2006 S49 Scorpio et al., 2007 S50Smith, 2006 S51 Silver, 2006 S52 Kotnik et al., 2007 S53 Katz &Caufield, 2003 S54 Zoeiby et al., 2003 S55 de Kruijff et al., 2008 S56Kimura & Bugg, 2003 S57 Hilpert et al., 1981 S58 Namboori & Graham, 2008S59 Hartmann & König, 1990 S60 Guo et al., 2007 S61 Scholte et al., 2004S62 Hammes et al., 1979 S63 Kandler & König, 1998 S64 Bouhss et al.,2008 S65 Ruiz, 2008 S66 Lindahl & Chang, 2001 S67 Musfeldt & Schönheit,2002 S68 Eggen et al., 1991 S69 Ragsdale, 2003 S70 Dermouni & Ansorg,2001 S71 Ansorg et al., 2003 S72 Bock et al., 1996 S73 Lin et al., 2003S74 Lin & Whitman, 2004 S75 Kato et al., 2006 S76 Grochowski et al.,2005 S77 Grochowski & White, 2008 S78 Kadziola et al., 2005 S79Martinez-Cruz et al., 2002 S80 Goenrich et al., 2005 S81 Werken van deet al., 2008 S82 Soderberg, 2005 S83 Lee et al., 2001 S84 Sprott et al.,1979 S85 Storer et al., 1981 S86 Thompson et al., 1998 S87 Mukhopadhyayet al., 1998 S88 Mukhopadhyay et al., 2000 S89 Mukhopadhyay et al., 2001S90 Shieh & Whitman, 1987 S91 Bobik & Wolfe, 1989 S92 Heim et al., 1998S93 Lemker et al., 2001 S94 Lemker et al., 2003 S95 Lewalter & Müller,2006 S96 Schäfer et al., 2006 S97 Schäfer et al., 2006 S98 Coskun etal., 2002 S99 Coskun et al., 2004 S100 Lingl et al., 2003 S101 Sprott &Jarrell, 1982 S102 Grüber & Marshansky, 2008 S103 Pisa et al., 2007 S104Müller et al., 1999 S105 Müller et al., 2004 S106 Ferry, 1999 S107 Alexet al., 1990 S108 Tersteegen & Hedderich, 1999 S109 Anderson et al.,2009 S110 Porat et al., 2006 S111 Woo et al., 1993 S112 Shah & Clark,1990 S113 Stojanowic et al., 2003 S114 Shima et al., 2002 S115 Thauer etal., 1993 S116 Aufhammer et al., 2005 S117 Hedderich et al., 2005 S118Mauer et al., 2002 S119 Deppenmeier, 2002 S120 Shokes et al., 2005 S121de Poorter et al., 2003 S122 Schäfer et al., 1999 S123 Pilak et al.,2006 S124 Shima et al., 2008 S125 Vignais et al., 2001 S126 Hendrickson& Leigh, 2008 S127 Klein et al., 1995 S128 Hagemeier et al., 2003 S129Mukhopadhyay & Daniels, 1989 S130 Mukhopadhyay et al., 1995 S131Jacobson et al., 1982 S132 Acharya et al., 2006 S133 Mamat et al., 2002S134 DiMarco et al., 1986 S135 Donnelly et al., 1985 S136 Vaupel et al.,1996 S137 Whitman & Wolfe, 1985 S138 Whitman & Wolfe, 1987 S139 Harmeret al., 2008 S140 Ermler, 2005 S141 Grabarse et al., 2001 S142 Selmer etal., 2000 S143 Ermler et al., 1997 S144 Prins et al., 1972 S145 Attwood& McSweeney, 2008 S146 Rospert et al., 1992 S147 Goenrich et al., 2004S148 Buckel & Golding, 2006 S149 Ellermann et al., 1988 S150 Sauer, 1991S151 Wackett et al., 1987 S152 Gottschalk & Thauer, 2001 S153 Kenealy &Zeikus, 1981 S154 Stupperich, 1993 S155 Becher et al., 1992 S156Andreesen & Makdessi, 2008 S157 Hochheimer et al., 1995 S158 Hochheimeret al., 1996 S159 Hochheimer et al., 1998 S160 Deppenmeier, 2002 S161Vorholt, 1997 S162 Wasserfallen, 1994 S163 Heath & Rock, 2004 S164 Heathet al., 2001 S165 Campbell & Cronan, 2001 S166 Payne et al., 2001 S167Payne, 2008 S168 Daiyasu et al., 2002 S169 Koga & Morii, 2007 S170Miller & Wolin, 2001 S171 Samuel et al., 2007 S172 de Ruyck & Wouters,2008 S173 Bonanno et al., 2001 S174 Friesen & Rodwell, 2004 S175 Istvan,2001 S176 Smit & Mushegian, 2000 S177 Boucher et al., 2004 S178 Barkleyet al., 2004 S179 Hoshino et al., 2006 S180 Wouters et al., 2004 S181Grochowski et al., 2004 S182 Payandeh et al., 2006 S183 Mareso &Schneewind, 2008 S184 Mareso et al., 2007 S185 Praetorius-Ibba & Ibba,2003 S186 Kim et al., 2003 S187 Tumbula et al., 1999 S188 Tumbula etal., 2000 S189 Sheppard et al., 2008 S190 Klipcan et al., 2008 S191Sheppard et al., 2008 S192 Schmitt et al., 2005 S193 Oshikane et al.,2006 S194 Ataide & Ibba, 2006 S195 Jenal et al., 1991 S196 Pohlmann &Brötz-Oesterhelt, 2004 S197 Ambrogelly et al., 2005 S198 Ahel et al.,2002 S199 Ahel et al., 2005 S200 Kim et al., 1998 S201 Yang et al., 2007S202 Graupner et al., 2002 S203 Zhang et al., 2008 S204 Bello et al.,2007 S205 Nyce & White, 1996 S206 Sarkar et al., 1977 S207 Hilpert etal., 1981 S208 {hacek over (S)}urín et al., 2007 S209 Hunter et al.,2009 S210 DiMarco et al., 1990 S211 Thauer & Bonacher, 1994 S212 Vermeijet al., 1997 S213 Pfaltz et al., 1987 S214 Schulz et al., 2006 S215Moser et al., 2002 S216 Gilles & Thauer, 1983 S217 Graham & White, 2002S218 Drevland et al., 2008 S219 White, 2001 S220 Howell et al., 1998S221 Howell et al., 2000 S222 Grochowski et al., 2009 S223 Grochowski etal., 2008 5224 Kengen et al., 1991 5225 Vermeij et al., 1994 S226Vermeij et al., 1995 S227 Li et al., 2003 S228 Kwang-Pil et al., 2001S229 Nocek et al., 2007 S230 Kwang-Pil et al, 2002 S231 Guerra-Lopez etal., 2007 S232 Graham et al., 2003 S233 Joerger et al., 2000 S234Schümperli et al., 2007 S235 Grochowski et al., 2006 S236 Nam Shin etal., 2007 S237 Forouhar et al., 2008 S238 Graupner et al., 2002 S239Wise et al., 2003 S240 Graham et al., 2002 S241 Graupner & White, 2001S242 Graupner et al., 2000 S243 Kezmarsky et al., 2005 S244 Dumitru etal., 2003 S245 Dumitru & Ragsdale, 2004 S246 Scott & Rasche, 2002 S247Chistoserdova et al., 1998 S248 Rasche & White, 1998 S249 Chistoserdovaet al., 2004 S250 Howell & White, 1997 S251 Grochowski et al., 2007 S252Schneider et al., 2000 S253 Chistoserdova et al., 2003 S254 Bauer etal., 2004 S255 Morrison et al., 2008 S256 Graham et al., 2002 S257Ungerfeld et al., 2004 S258 Ungerfeld et al., 2007 S259 Nagar-Anthal etal., 1996 S260 Fischer et al., 2004 S261 Fischer et al., 2005 S262Osterman et al., 2003 S263 Romisch-Margl et al., 2008 S264 Mashhhadi etal., 2008 S265 Ammelburg et al., 2009 S266 Konig et al., 1994 S267Kandler & Konig, 1978 S268 Perez-Bercoff et al., 2006 S269 Larkin etal., 2007 S270 Waterhouse et al., 20

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LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210260174A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1-66. (canceled)
 67. A vaccine composition comprising an adjuvant, and apolypeptide comprising the amino acid sequence of SEQ ID NO:6542. 68.The vaccine composition of claim 67, wherein the polypeptide comprises aconjugate or fusion molecule.
 69. The vaccine composition of claim 67,wherein the vaccine composition is an animal vaccine composition againsta methanogen.
 70. The vaccine composition of claim 69, wherein themethanogen is Methanobrevibacter ruminantium.
 71. The vaccinecomposition of claim 69, wherein the animal is a ruminant.
 72. Thevaccine composition of claim 71, wherein the ruminant is selected fromthe group consisting of cattle, sheep, goats, buffalo, moose, antelope,caribou, and deer.
 73. The vaccine composition of claim 67, wherein thevaccine composition is a ruminant vaccine composition for use inreducing methane emissions.
 74. A kit for reducing methanogen growth ormethane production in a ruminant comprising the vaccine composition ofclaim
 67. 75. A method of vaccinating an animal against a methanogen,comprising the step of administering to said animal, the vaccinecomposition according to claim
 67. 76. The method of claim 75, whereinthe methanogen is Methanobrevibacter ruminantium.
 77. The method ofclaim 75, wherein the animal is a ruminant.
 78. The method of claim 77,wherein the ruminant is selected from the group consisting of cattle,sheep, goats, buffalo, moose, antelope, caribou, and deer.
 79. A methodof reducing methane emissions from a ruminant, comprising vaccinatingthe ruminant against a methanogen according to claim 75.